Lettuce variety nun 08224 ltl

ABSTRACT

A new and distinct lettuce variety NUN 08224 LTL is disclosed, as well as seeds and plants and heads or leaves thereof. NUN 08224 LTL is a Batavia lettuce variety, comprising resistance to Downy Mildew (Bremia lactucae) Isolates B1:16-37EU, Nasonovia ribisnigri Biotype Nr:0, and Lettuce Mosaic Virus (LMV) Pathotype II.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.63/285,319 filed on Dec. 2, 2021, which is hereby incorporated byreference in its entirety.

FIELD OF DISCLOSURE

The disclosure relates to the field of plant breeding and, morespecifically, to the development of lettuce variety NUN 08224 LTL. Thedisclosure further relates to vegetative reproductions of lettucevariety NUN 08224 LTL, methods for tissue culture of lettuce variety NUN08224 LTL, and to phenotypic variants of lettuce variety NUN 08224 LTL.The disclosure also relates to progeny of lettuce variety NUN 08224 LTL,and the hybrid varieties obtained by crossing lettuce variety NUN 08224LTL as a parent line with plants of other varieties or parent lines.

BACKGROUND OF THE DISCLOSURE

The goal of vegetable breeding is to combine various desirable traits ina single variety. Such desirable traits may include greater yield,resistance to diseases, insects or other pests, tolerance to heat anddrought, better agronomic quality, higher nutritional value, enhancedgrowth rate, and improved shelf life.

The development of commercial lettuce cultivars or varieties requiresthe crossing of lettuce plants, and the evaluation of the crosses.Pedigree breeding and recurrent selection are examples of breedingmethods used to develop cultivars from breeding populations. Breedingprograms combine desirable traits from two or more varieties or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. The newcultivars are crossed with other varieties and the inbred lines orhybrids from these crosses are evaluated to determine which havecommercial potential.

All cultivated forms of lettuce belong to the highly polymorphic speciesLactuca sativa that is grown for its edible head and leaves. Lactucasativa is in the Asteraceae (Compositae) family. Lettuce is related tochicory, sunflower, aster, dandelion, artichoke, and chrysanthemum.L.sativa is one of about 300 species in the genus Lactuca. There aremany types of lettuce, and new types are constantly in development.Types of lettuce include Cutting/Leaf, Iceberg/Crisphead, Cos orRomaine, Batavian, Salinas Group, Latin, Butterhead, Great Lakes Group,Eastern (Ithaca) Group, Bibb, Vanguard Group, Multileaf, or Stemlettuce. Lettuce is typically consumed fresh and occasionally as acooked vegetable. It is popularly used in salads, wraps, and sandwiches.

Lettuce is available in the United States year-round although thegreatest supply is from May through October. For planting purposes, thelettuce season is typically divided into three categories, early, mid,and late, with the coastal areas planting from January to August, andthe desert regions planting from August to December. California andArizona are the two largest producers of lettuce in the United States.

Changes in lifestyle primarily due to increasing health awarenessresults to growing demand for healthy convenience food. Supermarkets,restaurants, catering firms, and convenience stores are constantlylooking for more colorful garnishing for sandwiches, wraps, andready-to-eat snacks such as salads. The changing food and consumertrends present opportunities for breeding companies to develop newvarieties with specific shapes of leaves, specific average size ofleaves, prominent color, glossiness, taste, and a wide variety oftexture. Other breeding objectives include disease or pest resistance,yield, prolonged shelf life, and suitability to climatic requirements.

SUMMARY OF VARIOUS ASPECTS OF THE DISCLOSURE

The disclosure provides for a lettuce variety NUN 08224 LTL, productsthereof, and methods of using the same. NUN 08224 LTL is a Batavialettuce variety and is suitable for growing in the open field.

In another aspect, the plant of lettuce variety NUN 08224 LTL, or partthereof, or progeny thereof comprises resistance to Downy Mildew (Bremialactucae) Isolates B1:16-37EU, Nasonovia ribisnigri Biotype Nr:0, andLettuce Mosaic Virus (LMV) Pathotype II, measured according to UPOVstandards described in TG/13/11.

The disclosure also provides a lettuce plant or part thereof having allof the physiological and morphological characteristics of the plant oflettuce variety NUN 08224 LTL when grown under the same environmentalconditions. The disclosure also provides for a progeny of lettucevariety NUN 08224 LTL. In one aspect, the disclosure provides a progenyretaining all or all but one, two, or three of the “distinguishingcharacteristics” of the plant of lettuce variety NUN 08224 LTL, or allbut one, two, or three of the “morphological and physiologicalcharacteristics” of the plant of lettuce variety NUN 08224 LTL andmethods of producing that plant or progeny.

In another aspect, the disclosure provides a plant or a progeny havingall of the physiological and morphological characteristics of the plantlettuce variety NUN 08224 LTL when grown under the same environmentalconditions. In another aspect, the plant or progeny has all or all butone, two, or three of the physiological and morphologicalcharacteristics of lettuce variety NUN 08224 LTL when grown under thesame environmental conditions and e.g., evaluated at significance levelsof 1%, 5%, or 10% significance (which can be expressed as a p-value) forquantitative characteristics and identical (same type or degree) fornon-quantitative characteristics, wherein a representative sample ofseed of variety NUN 08224 LTL has been deposited under Accession NumberNCIMB 43899. In another aspect, the plant or progeny has all or all butone, two, or three of the physiological and morphologicalcharacteristics as listed in Tables 1-3 of lettuce variety NUN 08224 LTLwhen measured under the same environmental conditions and e.g.,evaluated at significance levels of 1%, 5%, or 10% significance (whichcan also be expressed as a p value) for quantitative characteristics andidentical (same type or degree) for non-quantitative characteristics.

In another aspect, the plant of lettuce variety NUN 08224 LTL, or partthereof, or a progeny thereof has 16, 17, or more or all of thefollowing distinguishing characteristics when compared to the ReferenceVariety as shown in Table 4:

-   -   a. smaller plant spread of frame leaves;    -   b. shorter plant height;    -   c. lighter plant weight;    -   d. thinner diameter at base of head (core width);    -   e. medium oblate mature leaf shape;    -   f. medium intensity of green color of mature leaf;    -   g. weak glossiness of upper side of mature leaf;    -   h. weak to medium mature leaf blistering;    -   i. medium undulation of margin of mature leaf;    -   j. medium to dense incisions of margin of mature leaf;    -   k. absence of hue green of color of outer leaves;    -   l. shorter mature leaf length;    -   m. larger mature leaf width;    -   n. absent or weak auxiliary sprouting;    -   o. stronger bolting stem fasciation;    -   p. no resistance to Fusarium oxysporum sp. lactucae Race 1; and    -   q. resistant to Lettuce Mosaic Virus (LMV) Pathotype II, when        the numerical characteristics are determined at the 5%        significance level and determined by type or degree for        non-numerical characteristics for plants grown under the same        environmental conditions.

In another aspect, the disclosure provides a seed of lettuce variety NUN08224 LTL, wherein a representative sample of said seed has beendeposited under Accession Number NCIMB 43899. The disclosure alsoprovides for a plurality of seeds of lettuce variety NUN 08224 LTL. Thelettuce seed of variety NUN 08224 LTL may be provided as an essentiallyhomogeneous population of lettuce seed. The population of seed oflettuce variety NUN 08224 LTL may be particularly defined as anessentially free from other seed. The seed population may be grown intoplants to provide an essentially homogeneous population of lettuceplants described herein.

The disclosure also provides a plant grown from a seed of lettucevariety NUN 08224 LTL and plant part thereof.

The disclosure further provides a lettuce head and/or a lettuce leafproduced on a plant grown from a seed of lettuce variety NUN 08224 LTL.

The disclosure furthermore provides a seed growing or grown on a plantof lettuce variety NUN 08224 LTL (e.g., produced after pollination ofthe flower of lettuce variety NUN 08224 LTL).

In another aspect, the disclosure provides for a plant part obtainedfrom lettuce variety NUN 08224 LTL, wherein said plant part is: a leaf,a part of a leaf, a head, a part of a head, a fruit, a part of a fruit,pollen, an ovule, a cell, a petiole, a shoot or a part thereof, a stemor a part thereof, a root or a part thereof, a root tip, a stalk, acutting, a seed, a part of a seed, seed coat or another maternal tissuewhich is part of a seed grown on said variety, a hypocotyl, a cotyledon,a pistil, an anther, or a flower or a part thereof. Heads and leaves areparticularly important plant parts. Such plant parts may be suitable forsexual reproduction, vegetative reproduction, or a tissue culture. Inanother aspect, the plant part obtained from variety NUN 08224 LTL is acell, optionally a cell in a cell or tissue culture. That cell may begrown into a plant of lettuce variety NUN 08224 LTL.

In another aspect, the disclosure provides for an inbred variety of NUN08224 LTL.

In another aspect, the disclosure provides a cell culture of lettucevariety NUN 08224 LTL and a plant regenerated from lettuce variety NUN08224 LTL, wherein the plant has all of the characteristics of lettucevariety NUN 08224 LTL when grown under the same environmentalconditions, as well as methods for culturing and regenerating lettucevariety NUN 08224 LTL. Alternatively, a regenerated plant may have onecharacteristic that is different from lettuce variety NUN 08224 LTL, andwhich otherwise has all of the physiological and morphologicalcharacteristics of the plant of lettuce variety NUN 08224 LTL.

The disclosure also provides a vegetatively propagated plant of varietyNUN 08224 LTL having all or all but one, two, or three of themorphological and physiological characteristics of lettuce variety NUN08224 LTL, when grown under the same environmental conditions as well asmethods for vegetatively propagating lettuce variety NUN 08224.

In another aspect, the disclosure provides a method of producing alettuce plant comprising crossing lettuce variety NUN 08224 LTL withitself or another lettuce variety and selecting a progeny lettuce plantfrom said crossing or selfing.

The disclosure also provides a method of producing a lettuce plantderived from lettuce variety NUN 08224 LTL.

In a further aspect, the disclosure provides a method of producinghybrid lettuce seed comprising crossing a first parent lettuce plantwith a second parent lettuce plant and harvesting the resultant hybridlettuce seed, wherein said first parent lettuce plant or second parentlettuce plant is lettuce variety NUN 08224 LTL. Also provided is ahybrid lettuce seed produced from crossing a first parent lettuce plantwith a second parent lettuce plant and harvesting the resultant hybridlettuce seed, wherein said first parent lettuce plant or second parentlettuce plant is lettuce variety NUN 08224 LTL. Moreover, a hybridlettuce plant grown from the hybrid lettuce seed is provided.

In another aspect, the disclosure provides a method of introducing asingle locus conversion into the plant of variety NUN 08224 LTL, whereina representative sample of said seed has been deposited under AccessionNumber NCIMB 43899, wherein the plant comprises the single locusconversion and otherwise has all of the physiological and morphologicalcharacteristics of lettuce variety NUN 08224 LTL.

In yet another aspect, the disclosure provides a method of introducing adesired trait into the plant of lettuce variety NUN 08224 LTL, saidmethod comprises transforming the plant of variety NUN 08224 LTL, with atransgene that confers the desired trait, wherein the transformed plantcontains the desired trait and otherwise has all of the physiologicaland morphological characteristics of lettuce variety NUN 08224 LTL.

The disclosure also provides a method of producing a modified lettuceplant with a desired trait, wherein the method comprises mutating alettuce plant or plant part of lettuce variety NUN 08224 LTL, wherein arepresentative sample of said seed has been deposited under AccessionNumber NCIMB 43899, and wherein the mutated plant contains the desiredtrait and otherwise has all of the physiological and morphologicalcharacteristics of lettuce variety NUN 08224 LTL.

In one aspect, the single locus conversion or desired trait is yield,size, storage properties, color, taste, enhanced nutritional quality,post-harvest quality, male sterility, herbicide tolerance, insectresistance, pest resistance, disease resistance, environmental stresstolerance, modified carbohydrate metabolism, or modified proteinmetabolism, or the mutation occurs in any of the following genes:Ferulate-5-hydrxylase, dmr1, dmr6, NCED4, PAL, PPO.

In another aspect, the disclosure provides a container comprising theplant, plant part, or seed of lettuce variety NUN 08224 LTL.

Also provided is a food, a feed, or a processed product comprising theplant part of lettuce variety NUN 08224 LTL, wherein the plant part is aleaf or part thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the plant of lettuce variety NUN 08224 LTL taken from atrial in the Netherlands, 2020.

FIG. 2 shows the plant comparison between lettuce variety NUN 08224 LTLand the Reference Variety taken from a trial in the US, 2022.

FIG. 3 shows the cross section comparison between lettuce variety NUN08224 LTL and the Reference Variety taken from a trial in the US, 2022.

FIG. 4 shows the leaf comparison between lettuce variety NUN 08224 LTLand the Reference Variety taken from a trial in the US, 2022.

DEFINITIONS

“Lettuce” refers herein to plants of the species Lactuca sativa L. Themost commonly eaten parts of a lettuce plant are the head or a leaf. Thehead comprises a core and leaves, which may be divided in inner andouter leaves.

“Cultivated lettuce” refers to plants of Lactuca sativa (e.g.,varieties, breeding lines or cultivars of the species L. sativa as wellas crossbreds thereof, or crossbreds with other Lactuca sativa species,or even with other Lactuca species), cultivated by humans and havinggood agronomic characteristics.

The terms “lettuce plant designated NUN 08224 LTL,” “NUN 08224 LTL,”“inbred NUN 08224,” “08224 LTL,” “lettuce 08224” or “Bassari” are usedinterchangeably herein and refer to a lettuce plant of variety NUN 08224LTL, representative sample of said seed has been deposited underAccession Number NCIMB 43899.

A “seed of lettuce variety NUN 08224 LTL” refers to a lettuce seed whichcan be grown into a plant of lettuce variety NUN 08224 LTL, wherein arepresentative sample of viable seeds of lettuce variety NUN 08224 LTLhas been deposited under Accession Number NCIMB 43899. A seed can be inany stage of maturity, for example, a mature, viable seed, or animmature non-viable seed. A seed comprises an embryo and maternaltissues.

An “embryo of lettuce variety NUN 08224 LTL” refers to an embryo aspresent in a seed of lettuce variety NUN 08224 LTL, a representativesample of said seed of lettuce variety NUN 08224 LTL having beendeposited under Accession Number NCIMB 43899.

A “seed grown on lettuce variety NUN 08224 LTL” refers to a seed grownon a mature plant of variety NUN 08224 LTL or inside a fruit of lettucevariety NUN 08224 LTL. The “seed grown on lettuce variety NUN 08224 LTL”contains tissues and DNA of the maternal parent, lettuce variety NUN08224 LTL. The “seed grown on lettuce variety NUN 08224 LTL” contains anF1 embryo. When said seed is planted, it grows into a first generationprogeny plant of variety NUN 08224 LTL. Since lettuce variety NUN 08224LTL is an inbred variety and thus highly homozygous, the set ofchromosomes inherited by the first generation is predictable.

“Plant” includes the whole plant or any parts or derivatives thereof,preferably having the same genetic makeup as the plant from which it isobtained.

“Plant part” includes any part of a plant, such as a plant organ (e.g.,harvested or non-harvested fruits), a plant cell, a plant protoplast, aplant cell tissue culture or a tissue culture from which a whole plantcan be regenerated, a plant cell that is intact in a plant, a clone, amicropropagation, plant callus, a plant cell clump, a plant transplant,a vegetative propagation, a fruit, a harvested fruit, a part of a fruit,a leaf, a part of a leaf, pollen, an ovule, an embryo, a petiole, ashoot or a part thereof, a stem or a part thereof, a root or a partthereof, a root tip, a stalk, a cutting, a seed, a part of a seed, ahypocotyl, a cotyledon, a scion, a graft, a stock, a rootstock, apistil, an anther, or a flower. Seed can be mature or immature. Pollenor ovules may be viable or non-viable. Also, any developmental stage isincluded, such as seedlings, cuttings prior or after rooting, matureplants or leaves. Alternatively, a plant part may also include a plantseed which comprises one or two sets of chromosomes derived from theparent plant (e.g., from lettuce variety NUN 08224 LTL). An F1 progenyproduced from self-pollination of the inbred NUN 08224 LTL will thuscomprise two sets of chromosomes derived from lettuce variety NUN 08224LTL, while an F1 progeny derived from cross-fertilization of lettucevariety NUN 08224 LTL will comprise only one set of chromosomes fromlettuce variety NUN 08224 LTL and the other set of chromosomes from theother parent.

An “essentially homogeneous population of lettuce seed” is a populationof seeds where at least 77%, 98%, 99% or more of the total population ofseed are seed of lettuce variety NUN 08224 LTL.

An “essentially homogeneous population of lettuce plants” is apopulation of plants where at least 97%, 97%, 99% or more of the totalpopulation of plants are plants of lettuce variety NUN 08224 LTL.

The phrase “essentially free from other seed” refers to a population ofseeds where less than 3%, 2%, 1%, or even less, of the total populationof seed is seed that is not a lettuce seed or, in another option, lessthan 3%, 2%, 1%, or less, of the total population of seed is seed thatis not seed of lettuce variety NUN 08224 LTL.

“Cotyledon” refers to one of the first leaves of the embryo of a seedplant.

“Head” as used herein refers to lettuce heads, i.e., the plant withoutthe root system, for example, substantially all harvested leaves.Encompassed are immature leaves (e.g., “baby leaf”) and mature leaves.

The “base” of a plant is the part of a lettuce plant where the leavesare attached to the root system of the plant.

“Core length” of the internal lettuce stem is measured from the base ofthe cut and trimmed head to the tip of the stem.

“Core Length to Head Diameter Ratio (CLHD Ratio)” refers to the meancore length/head diameter ratio. It is calculated by dividing the meancore length with the mean head diameter. This is an indication of thehead shape and of the ability of a lettuce plant to reduce the amount ofsurface which is on or close to the ground.

“Head weight” refers to the mean weight of saleable lettuce head, cut,and trimmed to market specifications.

“Head diameter” refers to the mean diameter of the cut and trimmed head,sliced vertically, and measured at the widest point perpendicular to thestem.

“Head height” refers to the mean height of the cut and trimmed head,sliced vertically, and measured from the base of the cut stem to theleaf tip.

“Harvested plant material” refers herein to plant parts (e.g., leaves orheads detached from the whole plant) which have been collected forfurther storage and/or further use.

“Harvested seeds” refers to seeds harvested from a line or variety,e.g., produced after self-fertilization or cross-fertilization andcollected.

“First water date” refers to the date the seed first receives adequatemoisture to germinate. This can and often does equal the planting date.

“Maturity date” refers to the stage when the plants are of full size oroptimum weight, in marketable form or shape to be of commercial oreconomic value. This is also the time when measuring parameters of“mature” leaves. “Yield” means the total weight of all lettuce heads orleaves harvested per hectare of a particular line or variety. It isunderstood that “yield” expressed as weight of all lettuce heads orleaves harvested per hectare can be obtained by multiplying the numberof plants per hectare times the “yield per plant.”

“Marketable yield” means the total weight of all marketable lettuceheads or leaves harvested per hectare of a particular line or variety,e.g., lettuce heads or leaves suitable for being sold for freshconsumption, having good color, glossiness size and texture and no orvery low levels of deficiencies. A “marketable lettuce head or leaf” isa head or leaf that has commercial value.

“USDA descriptors” are the plant variety descriptors described forlettuce in the “Objective description of Variety—Lettuce (Lactuca sativaL.),” as published by U.S. Department of Agriculture, AgriculturalMarketing Service, Science and Technology, Plant Variety ProtectionOffice (June 2015, revised December 2018) and which can be downloadedfrom the world-wide web at ams.usda.gov/underservices/plant-variety-protection/pvpo-c-forms under lettuce. “Non-USDAdescriptors” are other descriptors suitable for describing lettuce.

“UPOV descriptors” are the plant variety descriptors described forlettuce in the “Guidelines for the Conduct of Tests for Distinctness,Uniformity and Stability,” TG/13/11 (Geneva 2006, last updated 2019 Jun.14), as published by UPOV (International Union for the Protection of NewVarieties and Plants) and which can be downloaded from the world-wideweb at upov.int/ under edocs/tgdocs/en/tg013.pdf, which is herebyincorporated by reference in its entirety. Likewise, “UPOV methods” todetermine specific parameters for the characterization of lettuce aredescribed at upov.int.

“RHS” or “RHS color” refers to the color chart of the RoyalHorticultural Society (UK), which publishes a botanical color chartquantitatively identifying colors according to a defined numberingsystem. The chart may be purchased from Royal Horticulture SocietyEnterprise Ltd RHS Garden; Wisley, Woking; Surrey GU236QB, UK, e.g., theRHS color chart 2007.

“Calibration Manual: DUS Test for Lettuce” refers to the calibrationbook for lettuce which provides guidance for describing a lettucevariety, as published by Naktuinbow (Netherlands) and NationalAgriculture and Food Research Organization (NARO Japan) (Dec. 26, 2016)and based on the UPOV Guideline TG/13/10 Rev. 2

“Reference Variety” refers herein to variety NUN 05404 LTL, a commercialvariety from Nunhems B.V. with commercial name Batraz, which has beenplanted in a trial together with lettuce variety NUN 08224 LTL. Thecharacteristics of lettuce variety NUN 08224 LTL were compared with thecharacteristics of the Reference Variety as shown in Tables 2 and 3. Thedistinguishing characteristics between lettuce variety NUN 08224 LTL andthe Reference Variety are shown in Table 4.

A plant having “all the physiological and morphological characteristics”of a referred-to-plant means a plant showing the physiological andmorphological characteristics of the referred-to-plant when grown underthe same environmental conditions, preferably in the same experiment;the referred-to-plant can be a plant from which it was derived, e.g.,the progenitor plant, the progenitor parent, the recurrent parent, theplant used for tissue- or cell culture, etc. A physiological ormorphological characteristic can be a numerical characteristic or anon-numerical characteristic. In one aspect, a plant has “all but one,two or three of the physiological and morphological characteristics” ofa referred-to-plant, or “all the physiological and morphologicalcharacteristics” of Tables 1-3 or “all or all but one, two or three ofthe physiological and morphological characteristics” of Tables 1-3.

The physiological and/or morphological characteristics mentioned aboveare commonly evaluated at significance levels of 1%, 5%, or 10% if theyare numerical (quantitative), or for having an identical degree (ortype) if not numerical (not quantitative), if measured under the sameenvironmental conditions. For example, a progeny plant or a single locusconverted plant or a mutated plant of lettuce variety NUN 08224 LTL mayhave one or more (or all) of the essential physiological and/ormorphological characteristics of said variety listed in Tables 1-3, asdetermined at the 5% significance level (i.e., p<0.05), when grown underthe same environmental conditions.

“Distinguishing characteristics” or “distinguishing morphological and/orphysiological characteristics” refers herein to the characteristicswhich distinguish a new variety from other lettuce varieties (i.e., aredifferent) when grown under the same environmental conditions. Thedistinguishing characteristics between lettuce variety NUN 08224 LTL andthe Reference variety are described in Table 4. When comparing lettucevariety NUN 08224 LTL with different varieties, the distinguishingcharacteristics may be different. In one aspect, the distinguishingcharacteristics may therefore include at least one, two, three or more(or all) of the characteristics listed in Tables 1-3. All numericaldistinguishing characteristics are statistically significantly differentat p<0.05 between lettuce variety NUN 08224 LTL and the other variety(e.g., the Reference Variety).

Lettuce variety NUN 08224 LTL has the following distinguishingcharacteristics when compared to the Reference Variety as presented inTable 4, when the numerical characteristics are determined at the 5%significance level and determined by type or degree for non-numericalcharacteristics for plants grown under the same environmentalconditions:

-   -   a. smaller plant spread of frame leaves;    -   b. shorter plant height;    -   c. lighter plant weight;    -   d. thinner diameter at base of head (core width);    -   e. medium oblate mature leaf shape;    -   f. medium intensity of green color of mature leaf;    -   g. weak glossiness of upper side of mature leaf;    -   h. weak to medium mature leaf blistering;    -   i. medium undulation of margin of mature leaf;    -   j. medium to dense incisions of margin of mature leaf;    -   k. absence of hue green of color of outer leaves;    -   l. shorter mature leaf length;    -   m. larger mature leaf width;    -   n. absent or weak auxiliary sprouting;    -   o. stronger bolting stem fasciation;    -   p. no resistance to Fusarium oxysporum sp. lactucae Race 1; and    -   q. resistant to Lettuce Mosaic Virus (LMV) Pathotype II.

Similarity and differences between two different plant lines orvarieties can be determined by comparing the number of morphologicaland/or physiological characteristics (e.g., characteristics as listed inTables 1-3) that are the same (i.e., statistically not significantlydifferent) or that are different (i.e., statistically significantlydifferent) between the two plant lines or varieties when grown under thesame environmental conditions. A numerical characteristic is consideredto be “the same” when the value for a numeric characteristic is notsignificantly different at the 1% (p<0.01) or 5% (p<0.05) significancelevel, using T-test, a standard method known to the skilled person.Non-numerical or “degree” or “type” characteristic is considered “thesame” when the values have the same “degree” or “type” when scored usingUSDA and/or UPOV descriptors, if the plants are grown under the sameenvironmental conditions.

In one aspect, a statistical of the quantitative characteristics showingthe degree of significance may be provided. Statistical significance isthe likelihood that a relationship between two or more variables iscaused by something other than chance, i.e., that the differences in themeans for quantitative characteristics of the plant of lettuce varietyNUN 08224 LTL, and the Reference Variety are significant or due tochance. For the purpose of proving differences or distinction betweenlettuce variety NUN 08224 LTL and the Reference Variety, a p-value of 5%or (0.05) or lower is considered statistically significant. This meansthat there is only a 5% probability that the observed result could havehappened just by chance or random variation.

The statistical analysis is drawn from a small sample of at least 15plants or plants of lettuce variety NUN 08224 LTL and the ReferenceVariety. Statistical points or parameters such as mean, minimum, median,maximum, and standard deviation are collected from the sample data toanalyze where the average is, how varied the data set is, and whetherthe data is skewed. For the purpose of determining whether the result ofthe data set is statistically significant, a T-Test is used, astatistical tool for proving significance in the means of the two groups(e.g., NUN 08224 LTL and the Reference Variety) at 5% significance level(a p-value of 5% or 0.05).

The term “traditional breeding techniques” encompasses herein crossing,selfing, selection, doubled haploid production, embryo rescue,protoplast fusion, marker assisted selection, mutation breeding etc. asknown to the breeder (e.g., methods other than geneticmodification/transformation/transgenic methods, e.g., gene editing), bywhich, for example, a genetically heritable trait can be transferredfrom one lettuce line or variety to another.

“Variety” or “cultivar” means a plant grouping within a single botanicaltaxon of the lowest known rank.

“Plant line” is, for example, an inbred variety or a breeding line whichcan be used to develop one or more varieties. Both are typically highlyhomozygous. Progeny obtained by selfing such a plant line has the samephenotype as its parents.

“Inbred variety” refers to an inbred (nearly homozygous) line or seedsthereof. For example, the (nearly homozygous) plant is self-pollinatedor the (nearly homozygous) female parent is pollinated with pollen ofthe same plant line to produce inbred seeds on the female parent.

“Progeny” as used herein refers to a plant obtained from a plantdesignated NUN 08224 LTL. A progeny may be obtained by regeneration ofcell culture or tissue culture or parts of a plant of said variety orselfing of a plant of said variety or by producing seeds of a plant ofsaid variety. In further aspects, progeny may also encompass plantsobtained from crossing of at least one plant of said variety withanother lettuce plant of the same or another variety or line, or wildlettuce plants. A progeny may comprise a mutation or a transgene. A“first generation progeny” is the progeny is directly derived from,obtained from, obtainable from or derivable from the parent plant by,e.g., traditional breeding methods (selfing and/or cross-pollinating) orregeneration (optionally combined with transformation or mutation).Thus, a plant of lettuce variety NUN 08224 LTL is the male parent, thefemale parent, or both of a first generation progeny of lettuce varietyNUN 08224 LTL. Progeny may have all of the physiological andmorphological characteristics of lettuce variety NUN 08224 LTL, whengrown under the same environmental conditions. Using methods such asbackcrossing, recurrent selection, mutation or transformation, one ormore specific characteristics may be introduced into said variety, or toa plant comprising all but one, two, or three of the morphological andphysiological characteristics of lettuce variety NUN 08224 LTL.

“Tissue Culture” refers to a composition comprising isolated cells ofthe same or a different type or a collection of such cells organizedinto parts of a plant. Tissue culture of various tissues of lettuce andregeneration of plants therefrom is well known and widely published(see, e.g., Teng et al., HortScience. 1992, 27(9): 1030-1032; Teng etal., HortScience. 1993, 28(6): 669-1671; Zhang et al., Journal ofGenetics and Breeding. 1992, 46(3): 287-290). Similarly, methods ofpreparing cell cultures are known in the art.

“Vegetative propagation,” “vegetative reproduction,” or “clonalpropagation” are used interchangeably herein and mean the method oftaking part of a plant and allowing that plant part to form at leastroots, and also refer to the plant or plantlet obtained by that method.Optionally, the vegetative propagation is grown into a mature plant.

“Regeneration” refers to the development of a plant from cell culture ortissue culture or vegetative propagation.

“Crossing” refers to the mating of two parent plants. The termencompasses “cross-pollination” and “selfing.”

“Selfing” refers to self-pollination of a plant, i.e., the transfer ofpollen from the anther to the stigma of the same plant.

“Cross-pollination” refers to the fertilization by the union of twogametes from different plants.

“Backcrossing” is a traditional breeding technique used to introduce atrait into a plant line or variety. The plant containing the trait iscalled the donor plant and the plant into which the trait is transferredis called the recurrent parent. An initial cross is made between thedonor parent and the recurrent parent to produce a progeny plant.Progeny plants which have the trait are then crossed to the recurrentparent. After several generations of backcrossing and/or selfing therecurrent parent comprises the trait of the donor. The plant generatedin this way may be referred to as a “single trait converted plant.” Thetechnique can also be used on a parental line of a hybrid.

The terms “gene converted,” “conversion plant,” or “single locusconverted plant” in this context refer to lettuce plants which aredeveloped by traditional backcrossing techniques, e.g., backcrossing, orvia genetic engineering (e.g., gene editing) or through mutationbreeding, wherein essentially all of the desired morphological andphysiological characteristics of parent variety or line are recovered,in addition to the one or more characteristics introduced into theparent via e.g., backcrossing technique (optionally including reversesynthesis of breeding line). It is understood that only the addition ofa further characteristic (e.g., addition of gene conferring a furthercharacteristic, such as a disease resistance gene), but also thereplacement/modification of an existing characteristic by a differentcharacteristic is encompassed herein (e.g., a mutant allele of a genecan modify the phenotype of a characteristic).

Likewise, a “Single Locus Converted (Conversion) Plant” refers to plantsdeveloped by plant breeding techniques comprising or consisting ofmutation breeding and/or by genetic transformation (e.g., gene editing)and/or by traditional breeding techniques, such as backcrossing, whereinessentially all of the desired morphological and physiologicalcharacteristics of a lettuce variety are recovered in addition to thecharacteristics of the single locus having been transferred into thevariety via the above-mentioned techniques, or wherein a morphologicaland physiological characteristic of the variety has beenreplaced/modified in the variety. In case of a hybrid, the gene may beintroduced, or modified, in the male or female parental line.

“Transgene” refers to a genetic locus comprising a DNA sequence whichhas been introduced into the genome of a lettuce plant bytransformation. A plant comprising a transgene stably integrated intoits genome is referred to as “transgenic plant.”

“Locus” (plural loci) refers to the specific location of a gene or DNAsequence on a chromosome. A locus may confer a specific trait.

“Genotype” refers to the genetic composition of a cell or organism.

“Allele” refers to one or more alternative forms of a gene locus. All ofthese loci relate to one trait. Sometimes, different alleles can resultin different observable phenotypic traits, such as differentpigmentation. However, many variations at the genetic level result inlittle or no observable variation.

As used herein, the terms “resistance” and “tolerance” are usedinterchangeably to describe plants that show no symptoms orsignificantly reduced symptoms to a specified biotic pest, pathogen,abiotic influence or environmental condition compared to a susceptibleplant. These terms are optionally also used to describe plants showingsome symptoms but that are still able to produce marketable product withan acceptable yield.

“Average” refers herein to the arithmetic mean.

The term “mean” refers to the arithmetic mean of several measurements.The mean, if not indicated otherwise within this application, refers tothe arithmetic mean of measurements on at least 15 different, randomlyselected plants of a variety or line.

DETAILED DESCRIPTION OF VARIOUS ASPECTS OF THE DISCLOSURE

The disclosure relates to the plant of lettuce variety NUN 08224 LTL,wherein a representative sample of seeds of said variety has beendeposited under Budapest Treaty, with Accession Number NCIMB 43899. NUN08224 LTL is a Batavia lettuce variety and is suitable for growing inthe open field.

In another aspect, the plant of lettuce variety NUN 08224 LTL, or partthereof, or progeny thereof comprises resistance to Downy Mildew (Bremialactucae) Isolates B1:16-37EU, Nasonovia ribisnigri Biotype Nr:0, andLettuce Mosaic Virus (LMV) Pathotype III, measured according to UPOVstandards described in TG/13/11.

The disclosure also provides a lettuce plant or part thereof having allof the physiological and morphological characteristics of the plant oflettuce variety NUN 08224 LTL, when grown under the same environmentalconditions.

The disclosure also provides a plant of lettuce variety NUN 08224 LTL,or part thereof, or a progeny plant thereof comprises all of themorphological and/or physiological characteristics (i.e., averagevalues, as indicated on the USDA Objective Description ofvariety—lettuce (unless indicated otherwise)) as shown in Tables 1-3,when grown under the same environmental conditions. A part of this plantis also provided.

The disclosure further provides a lettuce plant which does not differfrom the physiological and morphological characteristics of the plant oflettuce variety NUN 08224 LTL as determined at the 1%, 2%, 3%, 4%, or 5%significance level, when grown under the same environmental conditions.In a particular aspect, the plants are measured in the same trial (e.g.,the trial is conducted as recommended by the USDA or UPOV). Thedisclosure also comprises part of said plant, preferably a leaf or apart thereof.

The morphological and/or physiological differences between two differentindividual plants described herein (e.g., between lettuce variety NUN08224 LTL and a progeny of lettuce variety NUN 08224 LTL) or between aplant of lettuce variety NUN 08224 LTL or progeny of said variety, or aplant having all, or all but 1, 2, or 3 of the physiological andmorphological characteristics of lettuce variety NUN 08224 LTL (or all,or all but 1, 2, or 3 of the characteristics as listed in Tables 1-3)and another known variety can easily be established by growing saidvariety next to the each other or next to the other variety (e.g., inthe same field, under the same environmental conditions), preferably inseveral locations which are suitable for said lettuce cultivation, andmeasuring morphological and/or physiological characteristics of a numberof plants (e.g., to calculate an average value and to determine thevariation range/uniformity within the variety). For example, trials canbe carried out in Acampo, Calif., USA (N 38 degrees 07′261″/W 121degrees 18′ 807″, USA), whereby various characteristics, for example,maturity, leaf shape, size and texture, leaf color and glossiness, boltshape, surface and length, flower size and color, head weight, diseaseresistance, insect resistance, and resistance to physiological stresscan be measured and directly compared for species of lettuce.

Thus, the disclosure comprises a lettuce plant having one, two, or threeof the physiological and/or morphological characteristics which aredifferent from those of the plant of lettuce variety NUN 08224 LTL, andwhich otherwise has all of the physiological and morphologicalcharacteristics of the plant of lettuce variety NUN 08224 LTL e.g.,determined at 5% significance level for plants for numericalcharacteristics and determined by type/degree for non-numericalcharacteristics, when grown under the same environmental conditions. Inone aspect, the different characteristic(s) is/are a result of breedingwith lettuce variety NUN 08224 LTL and selection of a progeny plantcomprising one, two, or three characteristics which are different thanin lettuce variety NUN 08224 LTL. In another aspect, the differentcharacteristic is the result of a mutation (e.g., spontaneous mutationor a human induced mutation through e.g., targeted mutagenesis ortraditional mutagenesis such as chemically or radiation inducedmutagenesis), or it is the result of transformation.

The disclosure also relates to lettuce variety NUN 08224 LTL, which whencompared to the Reference Variety has the following distinguishingcharacteristics as shown in Table 4, when the numerical characteristicsare determined at the 5% significance level and determined by type ordegree for non-numerical characteristics for plants grown under the sameenvironmental conditions. Also encompassed are parts of that plant.

-   -   a. smaller plant spread of frame leaves;    -   b. shorter plant height;    -   c. lighter plant weight;    -   d. thinner diameter at base of head (core width);    -   e. medium oblate mature leaf shape;    -   f. medium intensity of green color of mature leaf;    -   g. weak glossiness of upper side of mature leaf;    -   h. weak to medium mature leaf blistering;    -   i. medium undulation of margin of mature leaf;    -   j. medium to dense incisions of margin of mature leaf;    -   k. absence of hue green of color of outer leaves;    -   l. shorter mature leaf length;    -   m. larger mature leaf width;    -   n. absent or weak auxiliary sprouting;    -   o. stronger bolting stem fasciation;    -   p. no resistance to Fusarium oxysporum sp. lactucae Race 1; and    -   q. resistant to Lettuce Mosaic Virus (LMV) Pathotype II.

The disclosure also relates to a seed of lettuce variety NUN 08224 LTL,wherein a representative sample of said seed has been deposited underthe Budapest Treaty with Accession Number NCIMB 43899.

In another aspect, a seed of hybrid variety NUN 08224 LTL is obtainableby crossing the male parent of lettuce variety NUN 08224 LTL with thefemale parent of lettuce variety NUN 08224 LTL, and harvesting the seedsproduced on the female parent. The resultant seed of said variety can begrown to produce plants of said variety.

In another aspect, the disclosure provides a plant grown from a seed oflettuce variety NUN 08224 LTL and a plant part thereof.

The disclosure also provides a lettuce head and/or a lettuce leafproduced on a plant grown from a seed of lettuce variety NUN 08224 LTL.

In another aspect, the disclosure provides for a plant part of lettucevariety NUN 08224 LTL, preferably a head or a leaf, a representativesample of seed from said variety has been deposited under AccessionNumber NCIMB 43899.

Also provided is a plant of lettuce variety NUN 08224 LTL, or a head ora leaf or other plant part thereof, produced from a seed, wherein arepresentative sample of said seeds has been deposited under theBudapest Treaty, with Accession Number NCIMB 43899.

Also provided is a plant part obtained from variety NUN 08224 LTL,wherein said plant part is a leaf, a part of a leaf, a head, a part of ahead, a fruit, a part of a fruit, pollen, an ovule, a cell, a petiole, ashoot or a part thereof, a stem or a part thereof, a root or a partthereof, a root tip, a cutting, a seed, a part of a seed, seed coat, oranother maternal tissue which is part of a seed grown on a said variety,a hypocotyl, a cotyledon, a pistil, an anther, or a flower or a partthereof. Such plant parts may be suitable for sexual reproduction (e.g.,pollen, a flower, an ovary, an ovule, an embryo, etc.), vegetativereproduction (e.g., a cutting, a root, a stem a cell, a protoplast, aleaf, a cotyledon, a meristem, etc.), or tissue culture (e.g., a leaf, apollen, an embryo, a cotyledon, a hypocotyl, a cell, a root, a root tip,an anther, a flower, a seed, a stem, etc.). Heads and leaves areparticularly important plant parts.

In a further aspect, the plant part obtained from variety NUN 08224 LTLis a cell, optionally a cell in a cell or tissue culture. The cell maybe grown into a plant of lettuce variety NUN 08224 LTL. A part oflettuce variety NUN 08224 LTL (or of a progeny of that variety or of aplant having all of the physiological and morphological characteristicsbut one, two, or three of lettuce variety NUN 08224 LTL) furtherencompasses any cells, tissues, organs obtainable from the seedlings orplants in any stage of maturity.

The disclosure also provides a tissue or cell culture comprisingregenerable cells of lettuce variety NUN 08224 LTL. Such tissue culturecan, for example, be grown on plates or in liquid culture or be frozenfor long term storage. The cells of lettuce variety NUN 08224 LTL, usedto start the culture can be any plant part suitable for vegetativereproduction, or, in a particular aspect, can be one or more of: anembryo, a meristem, a cotyledon, a hypocotyl, a pollen, a leaf, ananther, a root, a root tip, a petiole, a flower, a fruit, a seed, or astem. In another particular aspect, the tissue culture does not containsomaclonal variation or has reduced somaclonal variation. The skilledperson is familiar with methods to reduce or prevent somaclonalvariation, including regular reinitiation.

In another aspect, the disclosure provides a lettuce plant regeneratedfrom the tissue or cell culture of lettuce variety NUN 08224 LTL,wherein the regenerated plant is not different from lettuce variety NUN08224 LTL, in all, or all but one, two, or three, of the physiologicaland morphological characteristics, e.g., determined at 5% significancelevel for numerical characteristics and determined by type/degree fornon-numerical characteristics, when grown under the same environmentalconditions. Optionally, the plant has one, two, or three physiologicalor morphological characteristic that is different from lettuce varietyNUN 08224 LTL, wherein the difference or modification is effected bymutation or transformation with a transgene.

In another aspect, the disclosure provides a lettuce plant regeneratedfrom the tissue or cell culture of variety NUN 08224 LTL, wherein theplant has all or all but one, two or three of the physiological andmorphological characteristics, e.g., determined at 5% significance levelfor numerical characteristics and determined by type/degree fornon-numerical characteristics), when grown under the same environmentalconditions. Similarity or difference of a characteristic is determinedby measuring the characteristics of a representative number of plantsgrown under the same environmental conditions, determining whethertype/degree characteristics are the same and determining whethernumerical characteristics are different at the 5% significance level.

Lettuce variety NUN 08224 LTL, or its progeny, or a plant having allphysiological and/or morphological characteristics or all but one, two,or three which are different from those of lettuce variety NUN 08224LTL, can also be reproduced using vegetative reproduction methods.Therefore, the disclosure provides for a method of producing a plant orplant part of lettuce variety NUN 08224 LTL, comprising vegetativereproduction of lettuce variety NUN 08224 LTL. Vegetative propagationcomprises regenerating a whole plant from a plant part of lettucevariety NUN 08224 LTL, or from a progeny or from a plant having all ofthe physiological and morphological characteristics of said variety orall but one, two, or three different characteristics, such as a cutting,a cell culture, or a tissue culture.

The disclosure also provides methods of vegetatively propagating a partof the plant of variety NUN 08224 LTL. In certain aspects, the methodcomprises: (a) cultivating tissue or cells capable of being propagatedfrom lettuce variety NUN 08224 LTL to obtain proliferated shoots; and(b) rooting said proliferated shoots, to obtain rooted plantlets. Steps(a) and (b) may also be reversed, i.e., first cultivating said tissue toobtain roots and then cultivating the tissue to obtain shoots, therebyobtaining rooted plantlets. The rooted plantlets may then be furthergrown, to obtain plant. In one aspect, the method further comprises (c)growing plants from said rooted plantlets. Therefore, the method alsocomprises regenerating a whole plant from a part of lettuce variety NUN08224 LTL. In a particular aspect, the part to be propagated is acutting, a cell culture, or a tissue culture.

The disclosure also provides for a vegetatively propagated plant ofvariety NUN 08224 LTL (or from progeny of lettuce variety NUN 08224 LTL,or from a plant having all but one, two, or three of the physiologicaland/or morphological characteristics of lettuce variety NUN 08224 LTL),wherein the plant has all of the physiological and morphologicalcharacteristics of lettuce variety NUN 08224 LTL, e.g., determined at 5%significance level for numerical characteristics and determined by typeor degree for non-numerical characteristics, when grown under the sameenvironmental conditions. In another aspect, the propagated plant hasall but one, two, or three of the physiological and morphologicalcharacteristics of lettuce variety NUN 08224 LTL, e.g., determined at 5%significance level for numerical characteristics and determined bytype/degree for non-numerical characteristics, when grown under the sameenvironmental conditions. A part of said propagated plant or saidpropagated plant with one, two, or three differences is also provided.In another aspect, the propagated plant has all or all but one, two, orthree of the physiological and morphological characteristics of lettucevariety NUN 08224 LTL (e.g., as listed in Tables 1-3).

In another aspect, the disclosure provides a method for producing aplant part, preferably a head or a leaf, comprising growing a plant oflettuce variety NUN 08224 LTL until it develops at least one leaf ordevelops a head, and optionally collecting the head or leaf. Preferably,the head or leaf is collected at harvest maturity. In another aspect,the leaf is collected at baby leaf stage.

In another aspect, a plant of lettuce variety NUN 08224 LTL can beproduced by seeding directly in the soil (e.g., field) or by germinatingthe seeds in controlled environment conditions (e.g., greenhouses,hydroponic cultures, etc.) and optionally transplanting the seedlingsinto the field (see, e.g., Gonai et al., J. of Exp. Bot., 55(394): 111,2004; Turini, et. al., University of California Agricultural and NaturalResources, 2011, Publication 7215, 1-6; Smith, et. al., University ofCalifornia Agricultural and Natural Resources, 2011, Publication 7216,1-6). Lettuce may also be grown in tunnels. Moreover, said variety canbe grown in hydroponic cultures as described in, e.g., US 2008/0222949,which is herein incorporated by reference in its entirety, and theskilled person is familiar with various types of hydroponic cultures.Alternatively, seed of lettuce variety NUN 08224 LTL may be grown onpeat block for use as root ball lettuce. Furthermore, said variety maybe combined with 1, 2 or 3 different lettuce varieties to be grown as“composite lettuce” (see, e.g., EP 1197137, which is herein incorporatedby reference in its entirety).

In still another aspect, the disclosure provides a method of producing alettuce plant, comprising crossing a plant of lettuce variety NUN 08224LTL with a second lettuce plant at least once, allowing the seed todevelop and optionally harvesting said progeny seed. The skilled personcan select a progeny lettuce plant from said crossing. Optionally, theprogeny (grown from the progeny seed) is crossed twice, thrice, or four,six or seven times, and allowed to set seed. In one aspect, the first“crossing” further comprises planting seeds of a first and a secondparent lettuce plant, often in proximity so that pollination will occur;for example, mediated by insect vectors. Alternatively, pollen can betransferred manually. Where the plant is self-pollinated, pollinationmay occur without the need for direct human intervention other thanplant cultivation. After pollination, the plant can produce seed.

The disclosure also provides a method for collecting pollen of lettucevariety NUN 08224 LTL, comprising collecting pollen from a plant ofvariety NUN 08224 LTL. Alternatively, the method comprises growing aplant of lettuce variety NUN 08224 LTL until at least one flowercontains pollen and collecting the pollen. In a particular aspect, thepollen is collected when it is mature or ripe. A suitable method forcollecting pollen comprises collecting anthers or the part of the antherthat contains pollen, for example, by cutting the anther or the part ofthe anther off. Pollen can be collected in a container. Optionally,collected pollen can be used to pollinate a lettuce flower.

In yet another aspect, the disclosure provides a method of producing alettuce plant, comprising selfing a plant of variety NUN 08224 LTL oneor more times, and selecting a progeny plant from said selfing. In oneaspect, the progeny plant retains all or all but one, two, or three ofthe physiological and morphological characteristics of lettuce varietyNUN 08224 LTL, when grown under the same environmental conditions. In adifferent aspect, the progeny plant comprises all (or all but one, twoor three) of the physiological and morphological characteristic oflettuce variety NUN 08224 LTL of Tables 1-3.

The disclosure also provides a method for developing a lettuce plant ina lettuce breeding program, using lettuce variety NUN 08224 LTL, or itsparts as a source of plant breeding material. Suitable plant breedingtechniques are recurrent selection, backcrossing, pedigree breeding,mass selection, mutation breeding, genetic marker enhanced selection,and/or genetic transformation. In one aspect, the method comprisescrossing lettuce variety NUN 08224 LTL, or progeny of said variety, or aplant comprising all but 1, 2, or 3 or more of the morphological andphysiological characteristics of lettuce variety NUN 08224 LTL (e.g., aslisted in Tables 1-3) with a different lettuce plant, and wherein one ormore offspring of the crossing are subject to one or more plant breedingtechniques: recurrent selection, backcrossing, pedigree breeding, massselection, mutation breeding, genetic marker enhanced selection, genetictransformation (see, e.g., Brotman et al., Theor Appl Genet (2002)104:1055-1063). For breeding methods in general, see, e.g., Acquaah,Principles of Plant Genetics and Breeding, 2007, Blackwell Publishing,ISBN-13: 978-1-4051-3646-4.

In other aspects, the disclosure provides for a progeny plant of varietyNUN 08224 LTL, such as a progeny plant obtained by further breeding oflettuce variety NUN 08224 LTL. Further breeding with said varietyincludes selfing and/or cross-pollinating lettuce variety NUN 08224 LTLwith another lettuce plant or variety one or more times. In a particularaspect, the disclosure provides for a progeny plant that retains all ofthe morphological and physiological characteristics of lettuce varietyNUN 08224 LTL, optionally all or all but one, two, or threecharacteristics as listed in Tables 1-3, e.g., determined at 5%significance level for numerical characteristics and determined bytype/degree for non-numerical characteristics, when grown under the sameenvironmental conditions. In another aspect, the progeny is a firstgeneration progeny, i.e., the ovule or the pollen (or both) used in thecrossing is an ovule or pollen of lettuce variety NUN 08224 LTL, wherethe pollen comes from an anther of lettuce variety NUN 08224 LTL and theovule comes from an ovary of lettuce variety NUN 08224 LTL.

In another aspect, the plant and plant parts of lettuce variety NUN08224 LTL and progeny of said variety is provided, e.g., grown fromseeds, produced by sexual or vegetative reproduction, regenerated fromthe above-described plant parts, or regenerated from cell or tissueculture of the lettuce variety NUN 08224 LTL, in which the reproduced(seed propagated or vegetatively propagated) plant has all of thephysiological and morphological characteristics of lettuce variety NUN08224 LTL, e.g., as listed in Tables 1-3. In one aspect, said progeny oflettuce variety NUN 08224 LTL can be modified in one, two, or threecharacteristics, in which the modification is a result of mutagenesis ortransformation with a transgene.

In one aspect, pedigree selection is used as a breeding method fordeveloping a lettuce variety. Pedigree selection is also known as the“Vilmorin System of Selection,” see, e.g., Allard, John Wiley & Sons,Inc., 1999, 64-67. In general, selection is first practiced among F2plants. In the next season, the most desirable F3 lines are firstidentified, then desirable F3 plants within each line are selected. Thefollowing season and in all subsequent generations of inbreeding, themost desirable families are identified first, then desirable lineswithin the selected families are chosen. A family refers to lines thatwere derived from plants selected from the same progeny from thepreceding generation.

Using pedigree method, two parents may be crossed using an emasculatedfemale and a pollen donor (male) to produce F1 offspring. Lettuce is anobligate self-pollination species, which means that pollen is shedbefore stigma emergence, assuring 100% self-fertilization. Therefore, inorder to optimize crossing, a method of misting may be used to wash thepollen off prior to fertilization to assure crossing or hybridization.The F1 may be self-pollinated to produce segregating F2 generation.Individual plants may then be selected which represent the desiredphenotype in each generation (F3, F4, F5, etc.) until the traits arehomozygous or fixed within a breeding population.

Thus, progeny in connection with pedigree selection are either thegeneration (seeds) produced from the first cross (F1) or selfing (51),or any further generation produced by crossing and/or selfing (F2, F3,F4, F5, F6, F7, etc.) and/or backcrossing (BC1, BC2, BC3, BC4, BC5, BC6,BC7, etc.) one or more selected plants of the F1 and/or S1 and/or BC1generation (or plants of any further generation, e.g., the F2) withanother lettuce plant (an/or with wild relative of lettuce).

In yet another aspect, the disclosure provides for a method of producinga new lettuce plant. The method comprises crossing lettuce variety NUN08224 LTL or a plant of comprising all but 1, 2, or 3 of themorphological and physiological characteristics of said lettuce variety(as listed in Tables 1-3), or a progeny plant thereof, either as male oras female parent, with a second lettuce plant (or a wild relative oflettuce) one or more times, and/or selfing a lettuce plant of varietyNUN 08224 LTL, or a progeny plant thereof, one or more times, andselecting progeny from said crossing and/or selfing. The second lettuceplant may, for example, be a line or variety of the species Lactucasativa, or other Lactuca species.

In a further aspect, lettuce variety NUN 08224 LTL is used in crosseswith other, different, lettuce varieties to produce first generation(F1) lettuce hybrid seeds and plants with superior characteristics. In aparticular aspect, the disclosure provides a method a producing a hybridlettuce seed comprising crossing a first parent lettuce plant with asecond parent lettuce plant and harvesting the resultant seed, in whichthe first parent lettuce plant or second parent lettuce plant is lettucevariety NUN 08224 LTL. Also provided is a hybrid lettuce seed producedfrom crossing a first parent lettuce plant with a second parent lettuceplant and harvesting the resultant hybrid lettuce seed, wherein saidfirst parent lettuce plant or second parent lettuce plant is lettucevariety NUN 08224 LTL. In a further aspect, the hybrid lettuce plantproduce from the hybrid lettuce seed is provided.

The morphological and physiological characteristics (and thedistinguishing characteristics) of lettuce variety NUN 08224 LTL, areprovided, for example, in Tables 1-3. Encompassed herein is also a plantobtainable from lettuce variety NUN 08224 LTL (e.g., by selfing and/orcrossing and/or backcrossing with said variety and/or progeny of saidvariety) comprising all or all but one, two or three of thephysiological and morphological characteristics of lettuce variety NUN08224 LTL, listed in Tables 1-3 as determined at the 5% significancelevel for numerical characteristics or identical for non-numericalcharacteristics when grown under the same environmental conditionsand/or comprising one or more (or all; or all except one, two or three)when grown under the same environmental conditions. The morphologicaland/or physiological characteristics may vary somewhat with variation inthe environment (e.g., temperature, light intensity, day length,humidity, soil, fertilizer use), which is why a comparison under thesame environmental conditions is preferred. Colors can best be measuredagainst the Royal Horticultural Society (RHS) Chart. Also, at-harvestand/or post-harvest characteristics of heads or leaves can be compared,such as cold storage holding quality, post-harvest leaf crispness andleaf browning or pinking after cutting can be measured using knownmethods.

In another aspect, a seed of inbred variety NUN 08224 LTL is obtainableby selfing the variety and harvesting the seeds produced. The resultantseeds can be grown to produce plants of said variety.

In still another aspect, the disclosure provides a method of producing aplant derived from lettuce variety NUN 08224 LTL, the method comprising:(a) preparing a progeny plant derived from lettuce variety NUN 08224 LTLby crossing thee plant of variety NUN 08224 LTL, either as a male orfemale parent with a second plant or selfing lettuce variety NUN 08224LTL, or vegetative reproduction of lettuce variety NUN 08224 LTL, and(b) collecting seeds from said crossing or selfing or regenerating awhole plant from the vegetative cell—or tissue culture. Also providedare seeds and/or plants obtained by this method. All plants producedusing lettuce variety NUN 08224 LTL, as a parent are within the scope ofthe disclosure, including plant parts derived from lettuce variety NUN08224 LTL.

In further aspects, the method comprises growing a progeny plant of asubsequent generation from said seed of a progeny plant of a subsequentgeneration and crossing the progeny plant of a subsequent generationwith itself or a second plant and repeating the steps for an additional3-10 generations to produce a plant derived from lettuce variety NUN08224 LTL. The plant derived from lettuce variety NUN 08224 LTL may bean inbred line and the aforementioned repeating crossing steps may bedefined as comprising sufficient inbreeding to produce the inbred line.By selecting plants having one or more desirable traits, a plant derivedfrom lettuce variety NUN 08224 LTL is obtained which has some of thedesirable traits of the line as well as potentially other selectedtraits.

The disclosure provides for methods of producing plants which retain allthe morphological and physiological characteristics of a plant describedherein. The disclosure also provides for methods of producing a plantcomprising all but 1, 2, or 3 or more of the morphological andphysiological characteristics of lettuce variety NUN 08224 LTL (e.g., aslisted in Tables 1-3), but which are still genetically closely relatedto said variety. The relatedness can, for example, be determined byfingerprinting techniques (e.g., making use of isozyme markers and/ormolecular markers such as Single-nucleotide polymorphism (SNP) markers,amplified fragment length polymorphism (AFLP) markers, microsatellites,minisatellites, Random Amplified Polymorphic DNA (RAPD) markers,restriction fragment length polymorphism (RFLP) markers and others). Aplant is “closely related” to lettuce variety NUN 08224 LTL if its DNAfingerprint is at least 80%, 90%, 95% or 98% identical to thefingerprint of lettuce variety NUN 08224 LTL. In a particular aspect,AFLP markers are used for DNA fingerprinting (see, e.g., Vos et al.1995, Nucleic Acid Research 23: 4407-4414). A closely related plant mayhave a Jaccard's Similarity index of at least about 0.95 or 0.96 or more(see, e.g., “Guidelines for the Handling of a Dispute on EssentialDerivation in Lettuce” atworldseed.org/wp-content/uploads/2015/10/GuidelinesEDV_Lettuce_2004.pdf). The disclosure also provides a plant and avariety obtained or selected by applying these methods on lettucevariety NUN 08224 LTL. Such a plant may be produced by crossing and/orselfing, or alternatively, a plant may simply be identified and selectedamongst plants of said variety, or progeny of said variety, e.g., byidentifying a variant within lettuce variety NUN 08224 LTL, or progenyof said variety (e.g., produced by selfing) which variant differs fromlettuce variety NUN 08224 LTL, in one, two, or three of themorphological and/or physiological characteristics (e.g.,characteristics listed in Tables 1-3). In one aspect, the disclosureprovides a plant of lettuce variety NUN 08224 LTL having a Jaccard'sSimilarity index with said variety of at least 0.95, 0.96, 0.97, 0.98 oreven at least 0.99.

In some aspects, the disclosure provides a lettuce plant comprisinggenomic DNA having at least 95%, 96%, 97%, 98%, or 99% sequence identitycompared to the genomic DNA sequence of a plant of lettuce variety NUN08224 LTL, as deposited under Accession Number NCIMB 43899. In someaspects, the lettuce plant further comprises all or all but one, two, orthree of the physiological and morphological characteristics of lettucevariety NUN 08224 LTL (e.g., as listed in Tables 1-3). In other aspects,the lettuce plant comprises the distinguishing characteristics oflettuce variety NUN 08224 LTL.

For the purpose of this disclosure, the “sequence identity” ornucleotide sequences, expressed as a percentage, refers to the number ofpositions in the two optimally aligned sequences which have identicalresidues (×100) divided by the number of positions compared. A gap,i.e., a position in the pairwise length where a residue is present inone sequence but not in the other, is regarded as a position withnon-identical residues. A pairwise global sequence alignment of twonucleotide sequences is found by aligning the two consequences over theentire length according to the Needleman and Wunsch global alignmentalgorithm described in Needleman and Wunsch, 1970, J. Mol. Bio.48(3):443-53. A full implementation of the Needleman-Wunsch globalalignment algorithm is found in the needle program in The EuropeanMolecular Biology Open Software (see, EMBOSS, Rice, et. al., Trends inGenetics, June 2000, 16(6):276-77).

In one aspect, a plant of lettuce variety NUN 08224 LTL may also bemutated (by e.g., irradiation, chemical mutagenesis, heat treatment,etc.) and mutated seeds or plants may be selected in order to change oneor more characteristics of said variety. Methods such as TILLING(Targeting Induced Local Lesions in Genomes) may be applied to lettucepopulations in order to identify mutants.

Similarly, lettuce variety NUN 08224 LTL may be transformed andregenerated, whereby one or more chimeric genes are introduced into thevariety or into a plant comprising all but one, two, or three of thephysiological and morphological characteristics (e.g., as listed inTables 1-3). Many useful traits can be introduced into lettuce varietyNUN 08224 LTL by e.g., crossing lettuce variety NUN 08224 LTL with atransgenic lettuce plant comprising a desired transgene, as well as bydirectly introducing a transgene into lettuce variety NUN 08224 LTL bygenetic transformation techniques.

Any pest or disease resistance genes may be introduced into lettucevariety NUN 08224 LTL, progeny of said variety, or into a plantcomprising all but 1, 2, or 3 or more of the morphological andphysiological characteristics of lettuce variety NUN 08224 LTL (e.g., aslisted in Tables 1-3). Resistance to one or more of the followingdiseases or pests may be introduced into the plant described herein:Rhizomonas suberifaciens (Corky root rot), Bremia lactucae (Downymildew), Erysiphe cichoracearum f. sp. lactucae (Powdery mildew),Sclerotinia minor and Sclerotinia sclerotiorum (Lettuce Drop),Pseudomonas spp. (Bacterial Soft Rot), Botrytis cinerea (Grey Mold),Verticillium dahlia (Verticillium Wilt), Xanthomonas spp. (BacterialLeaf Spot), Microdochium panattonianum (Anthracnose), Fusarium oxysporumf. sp. lactucae, Rhizoctonia solani (Bottom Rot), Cabbage Loopers,Lettuce Root Aphid, Myzus persicae (Green Peach Aphid), Liriomyza langei(Pea Leafminer), Liriomyza trifolii (Serpentine Leafminer), Liriomyzasativae (Vegetable Leafminer), Foxglove Aphid, Potato Aphid, BeetArmyworm, Bemisia argentifolii (Silver Whitefly), and/or Aster Yellows.Other resistance genes, against pathogenic viruses (e.g., MirafioriLettuce Big Vein Virus (LMBVV), Lettuce Infectious Yellows Virus (LIYV),Lettuce Mosaic Virus (LMV), Lettuce Necrotic Stunt Virus (LNSV),Cucumber Mosaic Virus (CMV), Tomato Bushy Stunt Virus (TBSV), TomatoSpotted Wilt Virus (TSWV), Turnip Mosaic Virus, Beet Western YellowsVirus (BWYV), Alfalfa mosaic virus (AMV)), fungi, bacteria, nematodes,insects or other pests may also be introduced. In one aspect, resistanceagainst Nasonovia ribisnigri biotype Nr:0 and/or Nr:1 maybe introducedinto the plant disclosed herein. Also, any resistances to physiologicalstresses may be introduced into the plant described herein, or progenythereof or into a plant comprising all but 1, 2, or 3 or more of themorphological and physiological characteristics of said plant (e.g., aslisted in Tables 1-3). Resistance against one or more of the followingmay be introduced into the plant described herein: Tip burn, Heat,Drought, Cold, Salt and/or Brown rob (Rib Discoloration/Rib Blight).

Genetic transformation may, therefore, be used to insert a selectedtransgene into the lettuce plants of the disclosure described herein ormay, alternatively, be used for the preparation of transgenic lettuceplants which can be used as a source of the transgene(s), which can beintroduced into lettuce variety NUN 08224 LTL by e.g., backcrossing. Agenetic trait which has been engineered into the genome of a particularlettuce plant may then be moved into the genome of another lettuce plant(e.g., another variety) using traditional breeding techniques which arewell known in the art. For example, backcrossing is commonly used tomove a transgene from a transformed lettuce variety into an alreadydeveloped lettuce variety and the resulting backcross conversion plantwill then comprise the transgene(s).

Any DNA sequences, whether from a different species or from the samespecies, which are inserted into the genome using transformation, arereferred to herein collectively as “transgenes.” A “transgene” alsoencompasses antisense, or sense and antisense sequences capable of genesilencing. Thus, the disclosure also relates to transgenic plants oflettuce variety NUN 08224 LTL. In some aspects, a transgenic plant oflettuce variety NUN 08224 LTL may contain at least one transgene butcould also contain at least 1, 2, 3, 4, or more transgenes.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to aregulatory element active in plant cells (e.g., promoter). Theexpression vector may contain one or more such operably linkedgene/regulatory element combinations. The vector may be in the form of aplasmid and can be used alone or in combination with other plasmids toprovide transformed lettuce plants using transformation methods toincorporate transgenes into the genetic material of the lettuceplant(s). Transformation can be carried out using standard methods, suchas Agrobacterium tumefaciens mediated transformation or biolistic,followed by selection of the transformed cells and regeneration intoplants.

Plants can also be genetically engineered, modified, or manipulated toexpress various phenotypes of horticultural interest. Through thetransformation of lettuce, the expression of genes can be altered toenhance disease resistance, insect resistance, herbicide resistance,stress tolerance, horticultural quality, and other traits.Transformation can also be used to insert DNA sequences which control orhelp control male sterility or fertility restoration. DNA sequencesnative to lettuce as well as non-native DNA sequences can be transformedinto lettuce and used to alter levels of native or non-native proteins.Various promoters, targeting sequences, enhancing sequences, and otherDNA sequences can be inserted into the genome for the purpose ofaltering the expression of proteins. Reduction of the activity ofspecific genes (also known as gene silencing, or gene suppression) isdesirable for several aspects of genetic engineering in plants.

Genome editing is another method recently developed to geneticallyengineer plants. Specific modification of chromosomal loci or targetedmutation can be done through sequence-specific nucleases (SSNs) byintroducing a targeted DNA double strand break in the locus to bealtered. Examples of SSNs that have been applied to plants are: fingernucleases (ZFNs), transcription activator-like effector nucleases(TALENs), engineered homing endonucleases or meganucleases, andclustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 (Cas9), see, e.g., Songstad, et.al., Critical Reviews in Plant Sciences, 2017, 36:1, 1-23.

Thus, the disclosure provides a method of producing a plant of lettucevariety NUN 08224 LTL having a desired trait, comprising mutating aplant or plant part of variety NUN 08224 LTL, and selecting the plantthe desired trait, wherein the mutated plant contains the desired traitand otherwise retains all of the physiological and morphologicalcharacteristics of said variety, optionally as described Tables 1-3, andwherein a representative sample of seed of variety NUN 08224 LTL hasbeen deposited under Accession Number NCIMB 43899. In a further aspect,the desired trait is yield, nutritional value, taste, color,crunchiness, male sterility, herbicide tolerance, insect resistance,pest resistance, disease resistance, environmental stress tolerance,modified carbohydrate metabolism, or modified protein metabolism, or themutation occurs in any of the following genes: Ferulate-5-hydroxylase,dmr1, dmr6, NCED4, PAL, PPO.

The disclosure also provides a method for inducing a mutation in lettucevariety NUN 08224 LTL, comprising:

-   -   a. exposing the seed, plant, plant part, or cell of lettuce        variety NUN 08224 LTL to a mutagenic compound or to radiation,        wherein a representative sample of seed of said variety has been        deposited under Accession Number NCIMB 43899;    -   b. selecting the seed, plant, or plant part or cell of lettuce        variety NUN 08224 LTL having a mutation; and    -   c. optionally growing and/or multiplying the seed, plant, plant        part, or cell of lettuce variety NUN 08224 LTL having the        mutation.

The disclosure also provides a method of producing a lettuce planthaving a desired trait, wherein the method comprises transforming thelettuce plant with a transgene that confers the desired trait, whereinthe transformed plant contains the desired trait and otherwise retainsall of the physiological and morphological characteristics of the plantof variety NUN 08224 LTL. Thus, a transgenic lettuce plant is providedwhich is produced by the method described above, wherein the transgenicplant comprises the desired trait and otherwise all of the physiologicaland morphological characteristics of the plant of variety NUN 08224 LTL.

In another aspect, the disclosure provides a method of producing aprogeny of plant of variety NUN 08224 LTL further comprising a desiredtrait, said method comprising transforming the plant of lettuce varietyNUN 08224 LTL with at least one transgene that confers the desired traitand/or crossing the plant of lettuce variety NUN 08224 LTL with atransgenic lettuce plant comprising a desired transgene so that thegenetic material of the progeny that resulted from the cross containsthe desired transgene(s). Also encompassed is the progeny produced bythis method.

A desired trait (e.g., gene(s) conferring pest or disease resistance,herbicide, fungicide or insecticide tolerance, etc.) can be introducedinto lettuce variety NUN 08224 LTL, or progeny of said variety, bytransforming said variety or progeny of said variety with a transgenethat confers the desired trait, wherein the transformed plant retainsall or all but one, two or three of the phenotypic and/or morphologicaland/or physiological characteristics of lettuce variety NUN 08224 LTL,or the progeny of said variety and contains the desired trait, whereinthe desired trait is yield, nutritional value, taste, color,crunchiness, male sterility, herbicide tolerance, insect resistance,pest resistance, disease resistance, environmental stress tolerance,modified carbohydrate metabolism, or modified protein metabolism or themutation occurs in any of the following genes: Ferulate-5-hydrxylase,dmr1, dmr6, NCED4, PAL, PPO. In a particular aspect, the specifictransgene may be any known in the art or listed herein, including, apolynucleotide sequence conferring resistance to imidazolinone,sulfonylurea, glyphosate, glufosinate, triazine, benzonitrile,cyclohexanedione, phenoxy proprionic acid and L-phosphinothricin or apolynucleotide conferring resistance to Rhizomonas suberifaciens (Corkyroot rot), Bremia lactucae (Downy mildew), Erysiphe cichoracearum f. sp.lactucae (Powdery mildew), Sclerotinia minor and Sclerotiniasclerotiorum (Lettuce Drop), Pseudomonas spp. (Bacterial Soft Rot),Botrytis cinerea (Grey Mold), Verticillium dahlia (Verticillium Wilt),Xanthomonas spp. (Bacterial Leaf Spot), Microdochium panattonianum(Anthracnose), Fusarium oxysporum f. sp. lactucae, Rhizoctonia solani(Bottom Rot), Cabbage Loopers, Lettuce Root Aphid, Myzus persicae (GreenPeach Aphid), Liriomyza langei (Pea Leafminer), Liriomyza trifolii(Serpentine Leafminer), Liriomyza sativae (Vegetable Leafminer),Foxglove Aphid, Potato Aphid, Beet Armyworm, Bemisia argentifolii(Silver Whitefly), and/or Aster Yellows. Other resistance genes, againstpathogenic viruses (e.g., Mirafiori Lettuce Big Vein Virus (LMBVV),Lettuce Infectious Yellows Virus (LIYV), Lettuce Mosaic Virus (LMV),Lettuce Necrotic Stunt Virus (LNSV), Cucumber Mosaic Virus (CMV), TomatoBushy Stunt Virus (TBSV), Tomato Spotted Wilt Virus (TSWV), TurnipMosaic Virus, Beet Western Yellows Virus (BWYV), Alfalfa mosaic virus(AMV)), fungi, bacteria, nematodes, insects or other pests may also beintroduced.

By crossing and/or selfing also (one or more), single traits may beintroduced into, or modified in, lettuce variety NUN 08224 LTL (e.g.,using backcrossing breeding schemes), while retaining the remainingmorphological and physiological characteristics of said variety and/orwhile retaining one or more or all distinguishing characteristics. Asingle trait converted plant may thereby be produced. For example,disease resistance genes may be introduced, genes responsible for one ormore quality traits, yield, etc. Both single genes (e.g., dominant orrecessive) and one or more QTLs (quantitative trait loci) may betransferred into lettuce variety NUN 08224 LTL by breeding with saidvariety.

In another aspect, the disclosure provides a method of introducing asingle locus conversion, a single trait conversion, or a desired traitinto lettuce variety NUN 08224 LTL, comprising introducing a singlelocus conversion, a single trait conversion, or a desired trait in atleast one of the parents of lettuce variety NUN 08224 LTL and crossingthe converted parent with the other parent of lettuce variety NUN 08224LTL to obtain seed of said variety.

In another aspect, the step of introducing a single locus conversion, asingle trait conversion, or a desired trait in at least one of theparent plants comprises:

-   -   a. crossing the parental line of lettuce variety NUN 08224 LTL        with a second lettuce plant comprising the single locus        conversion, the single trait conversion, or the desired trait;    -   b. selecting F1 progeny plants that contain the single locus        conversion, the single trait conversion, or the desired trait;    -   c. crossing said selected progeny plants of step b) with the        parental line of step a) to produce a backcross progeny plant;    -   d. selecting backcross progeny plants comprising the single        locus conversion, the single trait conversion or the desired        trait and otherwise all or all but one, two, or three of the        physiological and morphological characteristics of the parental        line of step a) to produce selected backcross progeny plants;        and    -   e. optionally repeating steps c) and d) one or more times in        succession to produce selected second, third, or fourth, or        higher backcross progeny plants comprising the single locus        conversion, the single trait conversion, or the desired trait        and otherwise all or all but one, two, or three of the        physiological and morphological characteristics of the parental        line of step a) to produce selected backcross progeny plants,        when grown in the same environmental conditions.

The disclosure further relates to plants obtained by this method.

Alternatively, a single trait converted plant or single locus convertedplant may be produced by:

-   -   a. obtaining a cell or tissue culture of cells of the parental        line of lettuce variety NUN 08224 LTL;    -   b. genetically transforming or mutating said cells;    -   c. growing the cells into a plant; and    -   d. optionally selecting a plant that contains the desired single        locus conversion, single trait conversion, or the desired trait.

In another aspect, the disclosure provides a method of introducing asingle locus conversion, a single trait conversion, or a desired traitinto lettuce variety NUN 08224 LTL, comprising:

-   -   a. obtaining a combination of parental lines of lettuce variety        NUN 08224 LTL, optionally through reverse synthesis of breeding        lines;    -   b. introducing a single locus conversion, a single trait        conversion, or a desired trait in at least one of the parents of        step a); and    -   c. crossing the converted parent with the other parent of        step a) to obtain seed of lettuce variety NUN 08224 LTL.

In any of the above methods, wherein the single locus conversionconcerns a trait, the trait may be yield or pest resistance, or diseaseresistance. In one aspect, the trait is disease resistance and theresistance is conferred to Rhizomonas suberifaciens (Corky root rot),Bremia lactucae (Downy mildew), Erysiphe cichoracearum f. sp. lactucae(Powdery mildew), Sclerotinia minor and Sclerotinia sclerotiorum(Lettuce Drop), Pseudomonas spp. (Bacterial Soft Rot), Botrytis cinerea(Grey Mold), Verticillium dahlia (Verticillium Wilt), Xanthomonas spp.(Bacterial Leaf Spot), Microdochium panattonianum (Anthracnose),Fusarium oxysporum f. sp. lactucae, Rhizoctonia solani (Bottom Rot),Cabbage Loopers, Lettuce Root Aphid, Myzus persicae (Green Peach Aphid),Liriomyza langei (Pea Leafminer), Liriomyza trifolii (SerpentineLeafminer), Liriomyza sativae (Vegetable Leafminer), Foxglove Aphid,Potato Aphid, Beet Armyworm, Bemisia argentifolii (Silver Whitefly),and/or Aster Yellows. Other resistance genes, against pathogenic viruses(e.g., Mirafiori Lettuce Big Vein Virus (LMBVV), Lettuce InfectiousYellows Virus (LIYV), Lettuce Mosaic Virus (LMV), Lettuce Necrotic StuntVirus (LNSV), Cucumber Mosaic Virus (CMV), Tomato Bushy Stunt Virus(TBSV), Tomato Spotted Wilt Virus (TSWV), Turnip Mosaic Virus, BeetWestern Yellows Virus (BWYV), Alfalfa mosaic virus (AMV)), fungi,bacteria, nematodes, insects or other pests may also be introduced. Inone aspect, resistance against Nasonovia ribisnigri biotype Nr:0 and/orNr:1 maybe introduced into the plant disclosed herein. Also, anyresistances to physiological stresses may be introduced into the plantdescribed herein, or progeny thereof or into a plant comprising all but1, 2, or 3 or more of the morphological and physiologicalcharacteristics of said plant (e.g., as listed in Tables 1-3).Resistance against one or more of the following may be introduced intothe plant described herein: Tip burn, Heat, Drought, Cold, Salt and/orBrown rob (Rib Discoloration/Rib Blight).

The disclosure also provides a plant having one, two, or threephysiological and/or morphological characteristics which are differentfrom those of lettuce variety NUN 08224 LTL and which otherwise has allof the physiological and morphological characteristics of said variety,wherein a representative sample of seed of said variety has beendeposited under Accession Number NCIMB 43899. In particular, variantswhich differ from lettuce variety NUN 08224 LTL in none, one, two, orthree of the characteristics mentioned in Tables 1-3 are encompassed.

The disclosure also provides a lettuce plant comprising at least a setof first set of the chromosomes of lettuce variety NUN 08224 LTL, asample of seed of said variety has been deposited under Accession NumberNCIMB 43899; optionally further comprising a single locus conversion ormutation, wherein said plant has essentially all of the morphologicaland physiological characteristics of the plant comprising at least afirst set of the chromosomes of said variety. In another aspect, thesingle locus conversion or mutation confers a trait, wherein the traitis yield, color, size, taste, crunchiness, enhance nutritional quality,post-harvest quality, male sterility, herbicide tolerance, insectresistance, pest resistance, disease resistance, environmental stresstolerance, modified carbohydrate metabolism, or modified proteinmetabolism.

In one aspect, the disclosure provides for a haploid plant and/or adoubled haploid plant of variety NUN 08224 LTL or a plant having all butone, two, or three physiological and/or morphological characteristics oflettuce variety NUN 08224 LTL, or progeny of said variety. Haploid anddoubled haploid (DH) plants can, for example, be produced by cell ortissue culture and chromosome doubling agents and regeneration into awhole plant. DH production chromosome doubling may be induced usingknown methods, such as colchicine treatment or the like. In one aspect,the method comprises inducing a cell or tissue culture with a chromosomedoubling agent and regenerating the cells or tissues into a whole plant.

In another aspect, the disclosure comprises a method for producingdoubled haploid cells of lettuce variety NUN 08224, comprising makingdoubles haploid cells from haploids cells from the plant or plant partof lettuce variety NUN 08224 LTL with a chromosome doubling agent, suchas colchicine treatment (see, e.g., Nikolova and Niemirowicz-Szczytt(1996) Acta Soc Bot Pol 65:311-317).

In another aspect, the disclosure provides for haploid plants and/ordoubled haploid plants derived from lettuce variety NUN 08224 LTL that,when combined, make a set of parents of lettuce variety NUN 08224 LTL.The haploid plant and/or the doubled haploid plant of variety NUN 08224LTL can be used in a method for generating parental lines of lettucevariety NUN 08224 LTL.

The disclosure also provides methods for determining the identity ofparental lines of the plant described herein, in particular the identityof the female line. US 2015/0126380, which is hereby incorporated byreference, relates to a non-destructive method for analyzing maternalDNA of a seed. In this method, the DNA is dislodged from the seed coatsurface and can be used to collect information on the genome of thematernal parent of the seed. This method for analyzing maternal DNA of aseed, comprises the steps of contacting a seed with a fluid to dislodgeDNA from the seed coat surface, and analyzing the DNA thus dislodgedfrom the seed coat surface using methods known in the art. The skilledperson is thus able to determine whether a seed has grown on a plant ofa plant of lettuce variety NUN 08224 LTL is a progeny of said variety,because the seed coat of the seed is a maternal tissue geneticallyidentical to lettuce variety NUN 08224 LTL. Since lettuce variety NUN08224 LTL is an inbred variety, with a very high degree of homozygosity,any F1 progeny will inherit the same, predictable, set of chromosomesfrom its parent. Thus, the skilled person will also be able to identifymaternal tissues of a seed grown on an F1 progeny of lettuce variety NUN08224 LTL using the methods described in US 2015/0126380. In anotherparticular aspect, the skilled person can determine the identity of thefemale parental line of lettuce variety NUN 08224 LTL by analyzing theseed coat of a seed of that variety. In another aspect, the skilledperson can determine whether a seed is grown on lettuce variety NUN08224 LTL.

Using methods known in the art such as “reverse synthesis of breedinglines” or “reverse breeding”, it is possible to produce parental linesfor a hybrid plant such as lettuce variety NUN 08224 LTL. A skilledperson can take any individual heterozygous plant (called a“phenotypically superior plant” in Example 2 of US 2015/0245570 herebyincorporated by reference in its entirety; lettuce variety NUN 08224 LTLis such plant) and generate a combination of parental lines (reversebreeding parental lines) that, when crossed, produce the variety NUN08224 LTL. It is not necessary that the reverse breeding parental linesare identical to the original parental lines. Such new breeding methodsare based on the segregation of individual alleles in the sporesproduced by a desired plant and/or in the progeny derived from theself-pollination of that desired plant, and on the subsequentidentification of suitable progeny plants in one generation, or in alimited number of inbred cycles. Such a method is known from US2015/0245570 or from Wijnker et al., Nature Protocols Volume: 9, Pages:761-772 (2014) DOI: doi:10.1038/nprot.2014.049. Thus, the disclosureprovides a method for producing parental lines for a hybrid organism(e.g., lettuce variety NUN 08224 LTL), comprising in one aspect: a)defining a set of genetic markers present in a heterozygous form (H) ina partially heterozygous starting organism; b) producing doubled haploidlines from spores of the starting organism; c) geneticallycharacterizing the doubled haploid lines thus obtained for the said setof genetic markers to determine whether they are present in a firsthomozygous form (A) or in a second homozygous form (B); and d) selectingat least one pair of doubled haploid lines that have complementaryalleles for at least a subset of the genetic markers, wherein eachmember of the pair is suitable as a parental line for the hybridorganism.

In another aspect, the method for producing parental lines for hybridorganisms, e.g., of lettuce variety NUN 08224 LTL, which when crossedreconstitute the genome of lettuce variety NUN 08224 LTL, comprising:

-   -   a. defining a set genetic a) markers that are present a        heterozygous form (H) in a partially heterozygous starting        organism;    -   b. producing at least one further generation from the starting        organism by self-pollination (e.g., F2 or F3 generation);    -   c. selecting at least one pair of progeny organisms in which at        least one genetic marker from the set is present in a        complementary homozygous form (B vs. A, or A vs. B); and        optionally repeating steps b) and c) until at least one pair of        progeny organisms that have complementary alleles for at least a        subset of the genetic markers has been selected as parental        lines for a hybrid.

The disclosure also relates to a method of producing a combination ofparental lines of a plant of variety NUN 08224 LTL, comprising makingdouble haploid cells from haploid cells form said plant or a seed ofthat plant; and optionally crossing these parental lines to produce andcollect seeds. In another aspect, the disclosure relates to acombination of parental lines produced by this method. In still anotheraspect, the combination of parental lines can be used to produce a seedor plant of variety NUN 08224 LTL, when these parental lines arecrossed. In still another aspect, the disclosure relates to acombination of a parental lines from which a seed or plant having allphysiological and/or morphological characteristics of lettuce varietyNUN 08224 LTL (when the characteristics are determined at the 5%significance level for plants grown under the same environmentalconditions).

The disclosure also provides a combination of parental lines, which whencrossed, produce a seed or plant having all of the physiological and/ormorphological characteristics of lettuce variety NUN 08224 LTL but one,two, or three which are different (when grown under the sameenvironmental conditions).

In another aspect, a combination of a male and a female parental line oflettuce variety NUN 08224 LTL can be generated by methods describedherein, for example, through reverse synthesis of breeding lines.

In another aspect, the disclosure provides a method of determining thegenotype of the plant of lettuce variety NUN 08224 LTL, comprising thestep of detecting in the genome (e.g., a sample of nucleic acids) of theplant at least a first polymorphism or an allele. The skilled person isfamiliar with many suitable methods of genotyping, detecting apolymorphism or detecting an allele including SNP (Single NucleotidePolymorphism) genotyping, restriction fragment length polymorphismidentification (RFLP) of genomic DNA, random amplified polymorphicdetection (RAPD) of genomic DNA, amplified fragment length polymorphismdetection (AFLPD), polymerase chain reaction (PCR), DNA sequencing,allele specific oligonucleotide (ASO) probes, and hybridization to DNAmicroarrays or beads. Alternatively, the entire genome could besequenced. The method may, in certain aspects, comprise detecting aplurality of polymorphisms in the genome of the plant, for example, byobtaining a sample of nucleic acid from a plant and detecting in saidnucleic acids a plurality of polymorphisms. The method may furthercomprise storing the results of the step of detecting the plurality ofpolymorphisms on a computer readable medium.

Also provided is a plant part obtainable from variety NUN 08224 LTL (orfrom progeny of said variety or from a plant having all or all but one,two or three physiological and/or morphological characteristics whichare different from those of lettuce variety NUN 08224 LTL) or from avegetatively propagated plant of variety NUN 08224 LTL (or from itsprogeny or from a plant having all but one, two or three physiologicaland/or morphological characteristics which are different from those oflettuce variety NUN 08224 LTL), wherein the plant part is a leaf, aharvested leaf, a part of a leaf, a head, a harvested head, a part of ahead, pollen, an ovule, a cell, a petiole, a shoot or a part thereof, astem or a part thereof, a root or a part thereof, a root tip, a cutting,a seed, a part of a seed, seed coat or another maternal tissue which ispart of a seed grown on lettuce variety NUN 08224 LTL, or a hypocotyl, acotyledon, a pistil, an anther, or a flower or a part thereof.

A part of lettuce variety NUN 08224 LTL (or of progeny of said varietyor of a plant having all physiological and/or morphologicalcharacteristics but one, two or three which are different from those ofsaid variety) encompasses any cells, tissues, organs obtainable from theseedlings or plants, such as but not limited to: a lettuce head or apart thereof, a leaf or a part thereof, a cutting, hypocotyl, cotyledon,seed coat, pollen and the like. Such parts can be stored and/orprocessed further.

The disclosure also provides for a food product, a feed product, or aprocessed product comprising or consisting of a plant part describedherein. Preferably, the plant part is a lettuce head or leaf or a partthereof and/or an extract from a leaf or another plant part describedherein comprising at least one cell of lettuce variety NUN 08224 LTL.The food or feed product may be fresh or processed, e.g., dried,grinded, powdered, pickled, chopped, cooked, roasted, in a sauce, in asandwich, pasted, pureed or concentrated, juiced, pickled, canned,steamed, boiled, fried, blanched and/or frozen, etc.

Such plant part of lettuce variety NUN 08224 LTL can be stored and/orprocessed further. The disclosure thus also provides for a food or feedproducts comprising one or more of such parts, such as canned, chopped,cooked, roasted, in a sauce, in a sandwich, pasted, pureed orconcentrated, juiced, frozen, dried, pickled, or powdered lettuce headsor leaves from lettuce variety NUN 08224 LTL, or from progeny of saidvariety, or from a derived variety, such as a plant having all or allbut one, two, or three of the physiological and/or morphologicalcharacteristics of lettuce variety NUN 08224 LTL.

The disclosure further provides for food or feed products comprisingpart, or part of progeny of lettuce variety NUN 08224 LTL, or part of aplant having all but one, two, or three of the physiological andmorphological characteristics of lettuce variety NUN 08224 LTLcomprising one or more of such parts, optionally processed (e.g.,canned, chopped, cooked, roasted, in a sauce, in a sandwich, pasted,pureed or concentrated, juiced, frozen, dried, pickled, or powdered).

In another aspect, the plant, plant part, or seed of lettuce variety NUN08224 LTL is inside or more containers. For example, the disclosureprovides containers such as cans, boxes, crates, bags, cartons, ModifiedAtmosphere Packaging Films (e.g., biodegradable films), etc. comprisinga plant or part of a plant (fresh and/or processed) or seed of lettucevariety NUN 08224 LTL. In a particular aspect, the container comprises aplurality of seeds of lettuce variety NUN 08224 LTL, or a plurality ofplant parts of lettuce variety NUN 08224. The seeds may be pelletedprior to packing (to form pills or pellets) and/or may be disinfected,primed, and/or treated with various compounds, such as seed coating orcrop protection compound. The seed produces a plant of lettuce varietyNUN 08224 LTL.

In another aspect, the disclosure provides for a lettuce head or leaf ofvariety NUN 08224 LTL, or a part of a head or leaf of said variety. Thehead or leaf can be in any stage of maturity, for example, immature ormature. In another aspect, the disclosure provides for a containercomprising or consisting of a plurality of harvested lettuce heads orleaves or parts of lettuce heads or leaves of said variety, or lettuceheads or leaves of progeny thereof, or lettuce heads or leaves of aderived variety.

Marketable lettuce heads or leaves are generally sorted by size andquality after harvest. Alternatively, the lettuce heads or leaves can besorted by leaf size, shape, texture, glossiness, or color.

All documents (e.g., patent publications) are herein incorporated byreference in their entirety, including the following cited references:

-   Naktuinbow and NARO, “Calibration Manual: DUS Test for Lettuce,”    world-wide web at naktuinbow.nl-   US Department of Agriculture, Agricultural Marketing Service,    “Objective Description of Variety—Lettuce (Lactuca sativa L.)”,    world wide web at    ams.usda.gov/services/plant-variety-protection/pvpo-c-forms, under    lettuce.-   UPOV, “Guidelines for the Conduct of Tests for Distinctness,    Uniformity and Stability”, TG/13/11 (Geneva 2006, last updated 2017    Apr. 5), world-wide web at upov.int under edocs/tgdocs/en/tg013.pdf.-   Bertier, L. D., et. al., “High-Resolution Analysis of the    Efficiency, Heritability, and Editing Outcomes of    CRISPR/Cas9-Induced Modifications of NCED4 in Lettuce (Lactuca    sativa),” G3: Genes, Genomes, Genetics, 2018, vol. 8, pp. 1513-1521.-   Brotman, Y., et. al., “Resistance Gene Homologues in Melon are    Linked to Genetic Loci Conferring Disease and Pest Resistance”,    Theor Appl Genet, 2002, vol. 104, pp. 1055-1063, DOI    10.1007/s00122-001-0808-x-   Gonai, T., et al., “Abscisic Acid in the Thermoinhibition of Lettuce    Seed Germination and Enhancement of its Catabolism by Gibberellin”,    Journal of Experimental Botany, 2004, vol. 55(394), pp. 111-118.-   Hunter, P. J., et. al., “Oxidative Discolouration in Whole-head and    Cut Lettuce: Biochemical and Environmental Influences on a Complex    Phenotype and Potential Breeding Strategies to Improve Shelf-life,”    Euphytica, 2017, vol. 213(180), DOI 10.1007/s10681-017-1964-7.-   Needleman, S. B., et. al., “A General Method Applicable to the    Search for Similarities in the Amino Acid Sequence of Two Proteins”,    Journal of Molecular Biology, 1970, vol. 48(3), pp.-   Nikolova, V., et. al., “Diploidization of Cucumber (Cucumis sativus    L.) Haploids by Colchini Treatment”, Acta Societas Botanicorum    Poloniae, 1996, vol. 65, pp. 311-317.-   Smith, et. al., “Leaf Lettuce Production in California,” 2011,    University of California Agricultural and Natural Resources,    Publication 7216, pp. 1-6.-   Songstad, D. D., et. al., “Genome Editing of Plants,” Critical    Reviews in Plant Sciences, vol. 36, no. 1, pp 1-23.-   Teng, W., et al., “Rapid Regeneration of Lettuce from Suspension    Culture”, HortScience, 1992, vol. 27(9), pp. 1030-1032.-   Teng, W., et al., “Regenerating Lettuce from Suspension Culture in a    2-Liter Bioreactor”, HortScience, 1993, vol. 28(6), pp. 669-671.-   Turini, et. al., “Iceberg Lettuce Production in California,” 2011,    University of California Agricultural and Natural Resources,    Publication 7215, pp. 1-6.-   Vos, P., et al., “AFLP: A New Technique for DNA Fingerprinting”,    Nucleic Acids Research, 1995, vol. 23(21), pp. 4407-4414.-   Wijnker, E., et al., “Hybrid Recreation by Reverse breeding in    Arabidopsis thaliana”, Nature Protocols, 2014, vol. 9, pp. 761-772.    DOI: doi: 10.1038/nprot.2014.049-   Zhang, X., et al., “Genotypic Effects on Tissue Culture Response of    Lettuce Cotyledons”, Journal of Genetics and Breeding, 1992, vol.    46, pp. 287-290.-   US 2008/0222949-   EP 1 197 137 A1-   US 2015/0126380-   WO 2017/144669-   WO 2008/092505-   U.S. Pat. No. 8,237,019

Development of Lettuce Variety NUN 08224 LTL

The inbred variety NUN 08224 LTL was developed from an initial crossbetween lettuce lines. The female and male parents were crossed toproduce seeds. After the cross, progeny was self-pollinated orbackcrossed, followed by pedigree selection and line selection. Lettucevariety NUN 08224 LTL can be propagated by seeds or vegetatively, or byregeneration of a tissue culture. The seeds of lettuce variety NUN 08224LTL can be grown to produce inbred plants and parts thereof (e.g.,lettuce heads and leaves).

The Applicant concluded that lettuce variety NUN 08224 LTL is uniformand stable. This has been established through evaluation ofhorticultural characteristics. Several seed production events resultedin no observable deviation in genetic stability.

Deposit Information

A total of 625 seeds of variety NUN 08224 LTL was made and acceptedaccording to the Budapest Treaty by Nunhems B.V. on Dec. 2, 2021 at theNCIMB Ltd., Ferguson Building, Craibstone Estate, Bucksburn, AberdeenAB21 9YA, United Kingdom (NCIMB). The deposit has been assignedAccession Number NCIMB 43899. A statement indicating the viability ofthe sample has been provided. A deposit of lettuce variety NUN 08224 LTLis also maintained at Nunhems B.V. The lot number for these seeds is30195002005.

The deposit will be maintained in NCIMB for a period of 30 years, or 5years after the most recent request, or for the enforceable life of thepatent whichever is longer and will be replaced if it ever becomesnonviable during that period. Access to the deposits will be availableduring the pendency of this application to persons determined by theDirector of the U.S. Patent Office to be entitled thereto upon request.Subject to 37 C.F.R. § 1.808(b), all restrictions imposed by thedepositor on the availability to the public of the deposited materialwill be irrevocably removed upon the granting of the patent. Applicantdoes not waive any rights granted under this patent on this applicationor under the Plant Variety Protection Act (7 U.S.C. § 2321 et seq.).Accordingly, the requirements of 37 CFR § 1.801-1.809 have beensatisfied.

Characteristics of Lettuce Variety NUN 08224 LTL

The most similar variety to NUN 08224 LTL is referred to as NUN 05404LTL, a commercial variety from Nunhems B.V. with the commercial nameBatraz.

In Table 1, the characteristics of lettuce variety NUN 08224 LTL isshown based on a trial in the Netherlands in 2020 under open fieldconditions. For numerical characteristics, averages were calculated. Fornon-numerical characteristics, the type/degree were determined.

In Tables 2 and 3, a comparison between lettuce variety NUN 08224 LTLand Reference Variety is shown based on a trial in the USA under openfield conditions. Trial location: Acampo, Calif., USA; Seeding date: May12, 2022; Transplanting date: Jun. 6, 2022; Harvesting date: Jul. 14,2022. In Table 4, the distinguishing characteristics between lettucevariety NUN 08224 LTL and the Reference Variety are shown.

One replication of 30 plants of each variety (i.e., application andreference varieties), from which at least 15 plants or plant parts wererandomly selected and were used to measure characteristics. Fornumerical characteristics, averages were calculated. For non-numericalcharacteristics, the type/degree were determined. Similarity anddifferences between two different plant lines or varieties can bedetermined by comparing the number of morphological and/or physiologicalcharacteristics (e.g., characteristics as listed in Tables 1-3) that arethe same (i.e., statistically not significantly different) or that aredifferent (i.e., statistically significantly different) between the twoplant lines or varieties when grown under the same environmentalconditions. A numerical characteristic is considered to be “the same”when the value for a numeric characteristic is not significantlydifferent at the 1% (p<0.01) or 5% (p<0.05) significance level, usingT-test, a standard method known to the skilled person. Non-numerical or“degree” or “type” characteristic is considered “the same” when thevalues have the same “degree” or “type” when scored using USDA and/orUPOV descriptors, if the plants are grown under the same environmentalconditions. In one aspect, a statistical analysis of the quantitativecharacteristics showing the degree of significance at 5% significancelevel is provided (see, Tables 5-11).

In another aspect, the disclosure provides a plant having all of thephysiological and morphological characteristics of lettuce variety NUN08224 LTL as presented in Tables 1-3 when grown under the sameenvironmental conditions, and wherein a representative sample of seed ofsaid lettuce variety has been deposited under Accession Number NCIMB43899.

TABLE 1 Characteristics of Lettuce Variety NUN 08224 LTL ApplicationVariety Characteristics (NUN 08224 LTL) Plant type: Batavia Seed: Color:Black white, yellow, brown, black Type of culture: Open field Seedling:Anthocyanin coloration: Absent or very weak absent, present Plant:Diameter: Medium to large Degree of overlapping of upper part of leaves:Absent or weak absent or weak, medium, strong Number of leaves: MediumLeaf: Leaf attitude: Semi-erect erect, semi-erect, prostate Number ofdivisions: Absent or very few absent or very few, few, medium, many,very many Leaf shape: Medium oblate triangular, lanceolate, mediumoblate, narrow oblate, circular, broad elliptic, medium elliptic, narrowelliptic, linear, broad obtrullate, obovate, oblanceolate Shape of tip:Rounded acute, obtuse, rounded, obcordate Shape in longitudinal section:Flat concave, flat, convex Anthocyanin coloration: Absent or very weakabsent or very weak, weak, medium, strong, very strong Leaf color: GreenGreen, yellowish green, greyish green Intensity of green color: Mediumvery light, light, medium, dark, very dark Glossiness of upper side:Weak absent or very weak, weak, medium, strong, very strong Leafthickness: Medium very thin, thin, medium, thick, very thick Blistering:Weak to medium Absent or very weak, weak, medium, strong, very strongSize of blisters: Small small, medium, large Undulation of margin:Medium absent or very weak, weak, medium, strong, very strong Type ofincisions of margin: Irregularly dentate Depth of incisions of margin:Shallow absent or very shallow, shallow, medium, deep, or very deepDepth of secondary incisions of margin: Very shallow to shallow shallow,medium, deep Density of incisions of margin: Medium to dense Leafvenation: Flabellate not flabellate, semi-flabellate, flabellateBolting: Time of beginning of bolting under long day Very lateconditions: very early, early, medium, late, very late Axillarysprouting: Absent or weak Absent or weak, medium, strong Bolting stemfasciation: Strong absent or very weak, weak, medium, strong, verystrong Resistances: Bremia lactucae Isolate Bl: 16 Present Bremialactucae Isolate Bl: 17 Present Bremia lactucae Isolate Bl: 18 PresentBremia lactucae Isolate Bl: 19 Present Bremia lactucae Isolate Bl: 20Present Bremia lactucae Isolate Bl: 21 Present Bremia lactucae IsolateBl: 22 Present Bremia lactucae Isolate Bl: 23 Present Bremia lactucaeIsolate Bl: 24 Present Bremia lactucae Isolate Bl: 25 Present Bremialactucae Isolate Bl: 26 Present Bremia lactucae Isolate Bl: 27 PresentBremia lactucae Isolate Bl: 28 Present Bremia lactucae Isolate Bl: 29Present Bremia lactucae Isolate Bl: 30 Present Bremia lactucae IsolateBl: 31 Present Bremia lactucae Isolate Bl: 32 Present Bremia lactucaeIsolate Bl: 33 Present Bremia lactucae Isolate Bl: 34 Present Bremialactucae Isolate Bl: 35 Present Bremia lactucae Isolate Bl: 36 PresentBremia lactucae Isolate Bl: 36 Present Nasonovia ribisnigri Biotype Nr:0 Present Lettuce Mosaic Virus (LMV) Present

TABLE 2 Characteristics of Lettuce Variety NUN 08224 LTL and theReference Variety (Exhibit C - USDA Descriptors) Application VarietyReference Variety Characteristics (NUN 08224 LTL) (NUN 05404 LTL) UPOVMorphology: Seed: Seed color: Black Black white, yellow, brown, blackPlant: Degree of overlapping of upper part of leaves: Absent or weakAbsent or weak absent or weak, medium, strong Number of leaves (only forvarieties with Medium Medium degree of overlapping of upper part ofleaves absent or weak): very few, very few to few, few, few to medium,medium, medium to many, many, many to very many, very many Mature Leaf:Leaf attitude: Semi-erect Semi-erect erect, erect to semi-erect,semi-erect, semi-erect to horizontal, horizontal Number of divisions:Absent or very few Absent or very few absent or very few, very few tofew, few, few to medium, medium, medium to many, many, many to verymany, very many Shape (only for varieties with number Medium oblateNarrow oblate of divisions absent or very few): triangular, lanceolate,medium oblate, narrow oblate, circular, broad, medium elliptic, narrowelliptic, linear, broad obtrullate, obovate, oblanceolate Shape of apex(only for varieties with number Rounded Rounded of divisions absent orvery few): acute, obtuse, rounded, obcordate Longitudinal section: FlatFlat concave, concave to flat, flat, flat to convex, convex Anthocyanincoloration: Absent or very weak Absent or very weak absent or very weak,very weak to weak, weak, weak to medium, medium, medium to strong,strong, strong to very strong, very strong Color: Green RHS 144A GreenRHS 144A green, yellowish green, greyish green Intensity of green color:Medium Light to medium very light, very light to light, light, light tomedium, medium, medium to dark, dark, dark to very dark, very darkGlossiness of upper side: Weak Weak to medium absent or very weak, veryweak to weak, weak, weak to medium, medium, medium to strong, strong,strong to very strong, very strong Thickness: Medium Medium very thin,thin, medium, thick, very thick, Blistering: Weak to medium Mediumabsent or very weak, very weak to weak, weak, weak to medium, medium,medium to strong, strong, strong to very strong, very strong Size ofblisters: Small Small very small, very small to small, small, small tomedium, medium, medium to large, large, large to very large, very largeUndulation of margin: Medium Weak to medium absent or very weak, veryweak to weak, weak, weak to medium, medium, medium to strong, strong,strong to very strong, very strong Type of incisions of margin:Irregularly dentate Irregularly dentate crenate, regularly dentate,irregularly dentate, bidentate, tridentate Depth of incisions of margin:Shallow Shallow absent or very shallow, very shallow to shallow,shallow, shallow to medium, medium, medium to deep, deep, deep to verydeep, very deep Depth of secondary incisions of Very shallow to shallowVery shallow to shallow margin (only varieties with type of incisions ofmargin irregularly dentate bi- or tri-dentate): very shallow, veryshallow to shallow, shallow, shallow to medium, medium, medium to deep,deep, deep to very deep, very deep Density of incisions of margin:Medium to dense Sparse very sparse, very sparse to sparse, sparse,sparse to medium, medium, medium to dense, dense, dense to very dense,very dense Venation: Flabellate Flabellate not flabellate,semi-flabellate, flabellate Harvest maturity: Time of harvest maturity:very early, very early to early, early, early to medium, medium, mediumto late, late, late to very late, very late Bolting: Time of beginningof bolting: Very late Very late very early, very early to early, early,early to medium, medium, medium to late, late, late to very late, verylate Auxiliary sprouting: Absent or weak Medium absent or very weak,weak, medium, strong, very strong Bolting stem: fasciation: Strong Veryweak to weak absent or very weak, very weak to weak, weak, weak tomedium, medium, medium to strong, strong, strong to very strong, strongResistances: Resistance to Bremia lactucae Isolate Bl: 16EU PresentPresent Resistance to Bremia lactucae Isolate Bl: 17EU Present PresentResistance to Bremia lactucae Isolate Bl: 18EU Present PresentResistance to Bremia lactucae Isolate Bl: 20EU Present PresentResistance to Bremia lactucae Isolate Bl: 21EU Present PresentResistance to Bremia lactucae Isolate Bl: 22EU Present PresentResistance to Bremia lactucae Isolate Bl: 23EU Present PresentResistance to Bremia lactucae Isolate Bl: 24EU Present PresentResistance to Bremia lactucae Isolate Bl: 25EU Present PresentResistance to Bremia lactucae Isolate Bl: 26EU Present PresentResistance to Bremia lactucae Isolate Bl: 27EU Present PresentResistance to Bremia lactucae Isolate Bl: 28EU Present PresentResistance to Bremia lactucae Isolate Bl: 29EU Present PresentResistance to Bremia lactucae Isolate Bl: 30EU Present PresentResistance to Bremia lactucae Isolate Bl: 31EU Present PresentResistance to Bremia lactucae Isolate Bl: 32EU Present PresentResistance to Bremia lactucae Isolate Bl: 33EU Present PresentResistance to Bremia lactucae Isolate Bl: 34EU Present PresentResistance to Bremia lactucae Isolate Bl: 35EU Present PresentResistance to Bremia lactucae Isolate Bl: 36EU Present PresentResistance to Bremia lactucae Isolate Bl: 37EU Present PresentResistance to Nasonovia ribisnigri (Nr) Biotype Nr: 0 Present PresentResistance to Fusarium oxysporum sp. lactucae Race 1 AbsentIntermediately resistant Resistance to Lettuce Mosaic Virus (LMV)Pathotype II Present Absent USDA Morphology: Plant type: Butterhead,Novita Type, Iceberg Type, Batavia Batavia Batavia Type, Friseed'Amerique Type, Lollo Type, Oakloaf Type, Multi-divided Type, FrilliceType, Cos or Romaine, Bibb/Gem, Stem, Cutting/Whole Leaf, Latin, Other(specify) Plant: Spread of frame leaves, cm: 22.80 cm 25.44 cm Number ofleaves 13.55 15.10

TABLE 3 Characteristics of Lettuce Variety NUN 08224 LTL and theReference Variety (Non-USDA Descriptors) Application Variety ReferenceVariety Characteristics (NUN 08224 LTL) (NUN 05404 LTL) Seedling:Anthocyanin coloration: Absent or very weak Absent or very weak absent,present Plant: Height, cm: 10.33 cm 13.53 cm Weight, g: 216.10 g 332.50g Core - diameter at base of head, mm (core width): 16.71 mm 18.76 mmCore - ratio of spread of frame leaves/core diameter: 0.081 0.080 Core -height from base of head to apex, mm (core length): 27.91 mm 31.64 mmCore - height from base of head to apex range: 15.43-38.63 18.81-41.93Plant firmness: Loose Loose Mature leaf: Length, mm: 124.43 mm 145.44 mmWidth, mm: 139.41 mm 130.81 mm Size: Small Small small, medium, largeLeaf trichomes: Absent (smooth) Absent (smooth) absent, present Incisionof margin on apical part: Absent Absent absent, present Hue of greencolor of outer leaves: Absent Yellowish absent, yellowish, greyish,reddish Leaf attitude at 10-12 stage: Semi-erect Semi-erect erect,semi-erect, prostrate Leaf blade divisions (at 10-12 stage): EntireEntire entire, lobed, divided Head: Head formation: No head No head nohead, open head, closed head

TABLE 4 Distinguishing Characteristics between Lettuce Variety NUN 08224LTL and the Reference Variety Application Variety Reference VarietyCharacteristics (NUN 08224 LTL) (NUN 05404 LTL) Plant: Spread of frameleaves, cm: 22.80 cm 25.44 cm Height, cm: 10.33 cm 13.53 cm Weight, g:216.10 g 332.50 g Core - diameter at base of head, mm (core width):16.71 mm 18.76 mm Mature Leaf: Shape (only for varieties with numberMedium oblate Narrow oblate of divisions absent or very few):triangular, lanceolate, medium oblate, narrow oblate, circular, broad,medium elliptic, narrow elliptic, linear, broad obtrullate, obovate,oblanceolate Intensity of green color: Medium Light to medium verylight, very light to light, light, light to medium, medium, medium todark, dark, dark to very dark, very dark Glossiness of upper side: WeakWeak to medium absent or very weak, very weak to weak, weak, weak tomedium, medium, medium to strong, strong, strong to very strong, verystrong Blistering: Weak to medium Medium absent or very weak, very weakto weak, weak, weak to medium, medium, medium to strong, strong, strongto very strong, very strong Undulation of margin: Medium Weak to mediumabsent or very weak, very weak to weak, weak, weak to medium, medium,medium to strong, strong, strong to very strong, very strong Density ofincisions of margin: Medium to dense Sparse very sparse, very sparse tosparse, sparse, sparse to medium, medium, medium to dense, dense, denseto very dense, very dense Hue of green color of outer leaves: AbsentYellowish absent, yellowish, greyish, reddish Length, mm: 124.43 mm145.44 mm Width, mm: 139.41 mm 130.81 mm Bolting: Auxiliary sprouting:Absent or weak Medium absent or very weak, weak, medium, strong, verystrong Bolting stem: fasciation: Strong Very weak to weak absent or veryweak, very weak to weak, weak, weak to medium, medium, medium to strong,strong, strong to very strong, strong Resistances: Resistance toFusarium oxysporum sp. lactucae Race 1 Absent Intermediately resistantResistance to Lettuce Mosaic Virus (LMV) Pathotype II Present Absent

The results of the T-Test show significant differences at 5%significance level between lettuce variety NUN 08224 LTL and theReference Variety for plant spread of frame leaves, plant height, plantweight, diameter at base of head (core width), mature leaf length, andmature leaf width, as shown in Tables 5-10.

Table 5 shows a significant difference at 5% significance level betweenlettuce variety NUN 08224 LTL and the Reference Variety (p<0.001) forplant height (cm) based on the results of the trial conducted in the USin 2022.

TABLE 5 Application Variety Reference Variety Statistical Parameter (NUN08224 LTL) (NUN 05404 LTL) Number of samples 20 20 Minimum 7.60 11.30Maximum 13.70 16.0 Median 10.45 13.65 Mean 10.33 13.53 Standarddeviation 1.52 1.43

Table 6 shows a significant difference at 5% significance level betweenlettuce variety NUN 08224 LTL and the Reference Variety (p=0.005) forplant spread of frame leaves (cm) based on the results of the trialconducted in the US in 2022.

TABLE 6 Application Variety Reference Variety Statistical Parameter (NUN08224 LTL) (NUN 05404 LTL) Number of samples 20 20 Minimum 19.70 20.30Maximum 28.10 31.30 Median 22.60 25.50 Mean 22.80 25.44 Standarddeviation 2.24 3.28

Table 7 shows a significant difference at 5% significance level betweenlettuce variety NUN 08224 LTL and the Reference Variety (p=0.006) forplant weight (g) based on the results of the trial conducted in the USin 2022.

TABLE 7 Application Variety Reference Variety Statistical Parameter (NUN08224 LTL) (NUN 05404 LTL) Number of samples 20 20 Minimum 80.0 86.0Maximum 392.0 654.0 Median 225.0 324.0 Mean 216.10 332.50 Standarddeviation 90.82 150.29

Table 8 shows a significant difference at 5% significance level betweenlettuce variety NUN 08224 LTL and the Reference Variety (p=0.037) fordiameter at base of head (core width, mm) based on the results of thetrial conducted in the US in 2022.

TABLE 8 Application Variety Reference Variety Statistical Parameter (NUN08224 LTL) (NUN 05404 LTL) Number of samples 20 20 Minimum 12.51 12.89Maximum 22.71 24.15 Median 16.29 18.76 Mean 16.71 18.76 Standarddeviation 2.66 3.28

Table 9 shows a significant difference at 5% significance level betweenlettuce variety NUN 08224 LTL and the Reference Variety (p<0.001) formature leaf length (mm) based on the results of the trial conducted inthe US in 2022.

TABLEd Application Variety Reference Variety Statistical Parameter (NUN08224 LTL) (NUN 05404 LTL) Number of samples 20 20 Minimum 103.06 120.78Maximum 138.67 173.49 Median 126.06 139.98 Mean 124.43 145.44 Standarddeviation 10.05 16.18

Table 10 shows a significant difference at 5% significance level betweenlettuce variety NUN 08224 LTL and the Reference Variety (p=0.032) formature leaf width (mm) based on the results of the trial conducted inthe US in 2021-2022.

TABLE 10 Application Variety Reference Variety Statistical Parameter(NUN 08224 LTL) (NUN 05404 LTL) Number of samples 20 20 Minimum 115.36114.20 Maximum 158.18 152.05 Median 140.52 128.94 Mean 139.41 130.81Standard deviation 13.38 10.89

The results of the T-Test show no significant differences at 5%significance level between lettuce variety NUN 08224 LTL and theReference Variety height from base of head to apex (core length) asshown in Table 11.

Table 11 shows no significant difference at 5% significance levelbetween lettuce variety NUN 08224 LTL and the Reference Variety(p=0.076) for height from base of head to apex (core length, mm) basedon the results of the trial conducted in the US in 2022.

TABLE 11 Application Variety Reference Variety Statistical Parameter(NUN 08224 LTL) (NUN 05404 LTL) Number of samples 20 20 Minimum 15.4318.81 Maximum 38.63 41.93 Median 28.23 31.62 Mean 27.91 31.64 Standarddeviation 6.52 6.41

1. A plant, plant part, or seed of lettuce variety NUN 08224 LTL,wherein a representative sample of seed of said variety has beendeposited under Accession Number NCIMB
 43899. 2. The plant part of claim1, wherein the plant part is a head, a leaf, pollen, an ovule, a fruit,a scion, a rootstock, a cutting, a flower, or a cell.
 3. A seed thatproduces the plant of claim
 1. 4. A seed grown from the plant ofclaim
 1. 5. A lettuce plant grown from the seed of claim
 4. 6. A lettuceplant, or a part thereof, having all the physiological and morphologicalcharacteristics of the plant of claim 1 when grown under the sameenvironmental conditions.
 7. A tissue culture or cell culture ofregenerable cells of the plant or plant part of claim
 1. 8. The tissueculture or cell culture according to claim 7, comprising cells orprotoplasts derived from a plant part suitable for vegetativereproduction, wherein the plant part is an embryo, a meristem, acotyledon, a hypocotyl, a pollen, a leaf, an anther, a root, a root tip,a pistil, a petiole, a flower, a fruit, a seed, a stem, or a stalk.
 9. Alettuce plant regenerated from the tissue culture or cell culture ofclaim 7, wherein the plant has all of the physiological andmorphological characteristics of the plant of lettuce variety NUN 08224LTL when grown under the same environmental conditions, and wherein arepresentative sample of seed of said lettuce variety has been depositedunder Accession Number NCIMB
 43899. 10. A method of producing of theplant of claim 1, said method comprising vegetative propagating of atleast a part of the plant of lettuce variety NUN 08224 LTL, wherein arepresentative sample of seed of said lettuce variety has been depositedunder Accession Number NCIMB
 43899. 11. The method of claim 10, whereinsaid vegetative propagation comprises regenerating a whole plant from apart of lettuce variety NUN 08224 LTL, wherein a representative sampleof seed of said lettuce variety has been deposited under AccessionNumber NCIMB
 43899. 12. The method of claim 10, wherein said part is acutting, a cell culture, or a tissue culture.
 13. A vegetativelypropagated plant, or part thereof, produced by the method of claim 10,wherein the vegetatively propagated plant, or part thereof has all ofthe physiological and morphological characteristics of the plant oflettuce variety NUN 08224 LTL when grown under the same environmentalconditions, and wherein a representative sample of seed of said lettucevariety has been deposited under Accession Number NCIMB
 43899. 14. Amethod of producing a lettuce plant, said method comprising crossing theplant of claim 1 with itself or a second lettuce plant, selecting aprogeny lettuce plant from said crossing, allowing the progeny lettuceplant to form seed, and optionally repeating the crossing and selectingone or more times to produce further progeny seed.
 15. A progeny of thelettuce plant of claim 1, obtained by crossing the plant of lettucevariety NUN 08224 LTL with itself or another lettuce plant.
 16. A methodof producing an F1 hybrid lettuce seed, said method comprising crossinga first parent lettuce plant with a second parent lettuce plant andharvesting the resultant hybrid lettuce seed, wherein at the firstparent lettuce plant or second parent lettuce plant is the plant ofclaim
 1. 17. An F1 hybrid lettuce seed produced by the method of claim16.
 18. An F1 hybrid lettuce plant produced by growing the seed of claim17.
 19. A container comprising the plant, plant part, or seed ofclaim
 1. 20. A food, a feed, or a processed product comprising the plantpart of claim
 2. 21. A method of introducing a desired trait into theplant of claim 1, said method comprises transforming the plant oflettuce variety NUN 08224 LTL with a transgene that confers the desiredtrait, wherein a representative sample of seed of said lettuce varietyhas been deposited under Accession Number NCIMB 43899, and wherein thedesired trait is male sterility, herbicide tolerance, insect resistance,pest resistance, disease resistance, environmental stress tolerance,modified carbohydrate metabolism, or modified protein metabolism
 22. Alettuce plant produced by the method of claim 21, wherein thetransformed plant contains the desired trait and otherwise has all ofthe morphological and physiological characteristics of the plant oflettuce variety NUN 08224 LTL.
 23. A method of introducing a singlelocus conversion into the plant of claim 1 comprising: a. crossing theplant of claim 1 with a second lettuce plant comprising a desired singlelocus conversion to produce F1 progeny plants; b. selecting F1 progenyplants that have the single locus conversion to produce selected F1progeny plants; c. crossing the selected F1 progeny plants with lettucevariety NUN 08224 LTL to produce backcross progeny plants; d. selectingbackcross progeny plants that have the single locus conversion andotherwise comprise all of the physiological and morphologicalcharacteristics of lettuce variety NUN 08224 LTL to produce selectedbackcross progeny plants; and e. repeating steps (c) and (d) one or moretimes in succession to produce selected second or higher backcrossprogeny plants that comprise the single locus conversion and otherwisecomprise all of the physiological and morphological characteristics ofthe plant of lettuce variety NUN 08224 LTL, wherein a representativesample of seed of said lettuce variety has been deposited underAccession Number NCIMB
 43899. 24. The method of claim 23, wherein thesingle locus conversion confers male sterility, herbicide tolerance,insect resistance, pest resistance, disease resistance, environmentalstress tolerance, modified carbohydrate metabolism, or modified proteinmetabolism.
 25. A lettuce plant produced by the method of claim 23,wherein the plant further comprises the single locus conversion andotherwise has all of the morphological and physiological characteristicsof the plant of lettuce variety NUN 08224 LTL.
 26. A method of producinga modified lettuce plant, said method comprising mutating a target genein lettuce plant or plant part of lettuce variety NUN 08224 LTL, whereina representative sample of seed of said lettuce variety has beendeposited under Accession Number NCIMB
 43899. 27. The method of claim26, wherein the target gene modifies a desired trait and wherein thedesired trait is male sterility, herbicide tolerance, insect resistance,pest resistance, disease resistance, environmental stress tolerance,modified carbohydrate metabolism, or modified protein metabolism. 28.The method of claim 26, wherein the target gene is mutated by targetedgene editing.
 29. A lettuce plant produced by the method of claim 27,wherein the modified lettuce plant contains the desired trait andotherwise has all of the physiological and morphological characteristicsof lettuce variety NUN 08224 LTL.
 30. A method of producing doubledhaploid cells of the plant of claim 1, said method comprising makingdoubled haploid cells from haploid cells from the plant or seed oflettuce variety NUN 08224 LTL, wherein a representative sample of seedof said lettuce variety has been deposited under Accession Number NCIMB43899.
 31. A method for determining the genotype of the plant of claim1, said method comprising obtaining a sample of nucleic acids from saidplant and detecting in said nucleic acids a plurality of polymorphisms,thereby determining the genotype of the plant, and storing the resultsof detecting the plurality of polymorphisms on a computer readablemedium.
 32. A method of producing a lettuce head or a lettuce leaf, saidmethod comprising growing the plant of claim 1 until it develops atleast a leaf or head, and collecting the leaf or head.
 33. A lettucehead or a lettuce leaf produced by the method of claim
 32. 34. Acontainer comprising the lettuce head or lettuce leaf produced by themethod of claim
 32. 35. A method for developing a lettuce plant in alettuce breeding program, said method comprising applying plant breedingtechniques comprising recurrent selection, backcrossing, pedigreebreeding, mass selection, mutation breeding, genetic marker enhancedselection, or genetic transformation to the plant of claim 1 or partthereof, wherein said plant breeding techniques result in a developmentof a lettuce plant.
 36. A method of producing a lettuce plant derivedfrom the plant of claim 1 comprising: a. preparing a progeny lettuceplant derived from lettuce variety NUN 08224 LTL by crossing the plantof claim 1 with itself or with a second lettuce plant, wherein arepresentative sample of seed of said lettuce variety has been depositedunder Accession Number NCIMB 43899; b. crossing the progeny plant withitself or a second lettuce plant to produce seed of a progeny plant ofthe subsequent generation; c. growing a progeny plant of the subsequentgeneration from said seed and crossing the progeny plant of thesubsequent generation with itself or a second lettuce plant; and d.repeating step (b) and (c) for at least one more generation to produce alettuce plant derived from lettuce variety NUN 08224 LTL.